AN OVERVIEW OF GLUCURONIDATION IN DRUG METABOLIC …

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Rathod et al. World Journal of Pharmacy and Pharmaceutical Sciences

AN OVERVIEW OF GLUCURONIDATION IN DRUG METABOLIC

REACTIONS AND ITS METABOLITES IDENTIFICATION BY

CHROMATOGRAPHIC METHODS

Laximan Velip1, Srikanth P.

1, Sitesh Sah

1, Shraddha Jain

1, Surya Narayana P.

2,

Rajeshwari Rathod1*

Department of Pharmaceutical Analysis, Niper-Ahmedabad.

ABSTRACT

Metabolic transformation of the drugs is the critical factor in the drug

elimination from the body. One of such metabolic process is

glucuronide conjugation in the phase two reaction. Many scientists

have shown much interest in discussing the CYP 450 enzymatic

metabolism but the glucuronidation conjugation has its own

importance in conjugation metabolic reaction. Here we discussed about

all the uridine diphosphate glucuronosyl transferase (UGT) isoforms in

a detailed way and their location in the various parts of human body.

Glucuronidation are observed more frequently in moleculescontaining

functional groups like R-COOH, R-OH, R-NH2. Factors like age, gender, diet, smoking and

alcohol etc. will affect the glucuronidation conjugation reaction. To assess the

glucuronidation products various analysis is done like in-vitro tests of microsomal incubation

assays, sub cellular enzymatic assay, liquid chromatography mass spectroscopy, HRMS etc.,

are carried out. In presence of enzyme inducers and inhibitors, how UGTs will behave is

discussed here. Finally, the list of drugs that are undergoing glucuronidation metabolism and

their metabolites identification and characterization using chromatographic techniques are

mentioned.

KEY WORDS: Glucuronidation, UGT enzymes, HPLC, LC-MS/MS, GC-MS/MS.

1. INTRODUCTION

Metabolism is the most important phenomenon in the body for the elimination of drugs and

other endogenous substances. In these processes, the drug will undergo an enzymatical

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.421

Volume 8, Issue 6, 1492-1518 Review Article ISSN 2278 – 4357

Article Received on

18 April 2019,

Revised on 08 May 2019,

Accepted on 29 May 2019,

DOI: 10.20959/wjpps20196-14052

*Corresponding Author

Rajeshwari Rathod

Department of

Pharmaceutical Analysis,

Niper-Ahmedabad.


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bioconversion and form a new substance inside the human body (Krishna and Klotz 1994).

Liver is the major site for the metabolic process because the majority of the enzymes are

present in it. Generally, metabolic reactions are of three types: phase1, phase2, and phase 3

reactions. Phase 1 reactions are also called non-synthetic reactions and phase2 reactions are

called synthetic or conjugation reactions(King, Rios et al. 2000). where as in phase3

metabolic transporters like P-glycoprotein, multidrug resistance-associated protein and

organic anion transporting polypeptides are included, these all are energy dependent

transporters. By using these transporters compounds are exported from cells(Stanley 2017).

Drug metabolism is an important component in drug discovery and development since it

characterises the ADMET (absorption, distribution, metabolism, excretion and toxicology)

properties of new drug candidate. The main goal of drug metabolism studies is to find the

drug molecule behaviour after metabolism to give useful information that supports for better

drug design with adequate safety and use (Nnane and Tao 2005).

Glucuronidation is one of the process of drug metabolism and many other endogenous

compounds metabolism like bilirubin, androgens, oestrogens, mineralocorticoids,

glucocorticoids, fatty acid derivatives, retinoids and bile acids(Yang, Ge et al. 2017).

Glucuronidationcomes under the category of conjugation reactions because in

glucuronidation reaction a hydrophilic substance is attached to the drug molecule such as

glucuronic acid. After metabolism drug shows different types of actions i.e. conversion of the

active metabolite, conversion of inactive substance and conversion of a toxicsubstance from

pro-drug by different metabolicenzymes (Caldwell 1979).Glucuronic enzymes are

microsomal enzymes these are present in the endoplasmic reticulum of the liver, remaining

all are present cytosol(Zakim and Dannenberg 1992). For the elimination of many

xenobiotics and endobiotics, glucuronidation is the main pathway. In these process ethers,

esters, thiolic, N and C glucuronides are formed and greatly increases the water solubility in

each case with compared to parent compound. Glucuronides have a low volume of

distribution and protein binding so these are easily eliminated from body (Rowland, Miners et

al. 2013).

In metabolic process,glucuronidation is involved in huge number of compounds at distinctive

functional groups. Glucuronidation mediated by uridine 5-diphosphoglucuronic acid to

transfer glucuronide to one or other functional groups such as R-COOH, R-OH,R-NH2 etc.

Glucuronic acids links to O, S, N or C atoms and results in formation of glucuronides


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metabolite like N-glucuronide, O-glucuronide, S glucuronide and C-glucuronide etc. In case

of N-glucuronidation, it will occur in alkylamines, arylamines, hydroxylamines, carbonates,

thioureas and sulphonamides (Hawes 1998). In drugs like Amitriptyline, nicotine,

chlorpromazine, clozapine, doxepin, Imipramine, ketotifen, olanzapine etc.

C-Glucuronidation is likely to occur in humans where as it occurs very rarely for drugs like

sulfinpyrazone, feprazone, phenylbutazone, fenoprofen. The common feature involves in C-

glucuronidation is a pyrazolidinedionering having acidic carbon atoms (Nishiyama, Kobori et

al. 2006).

In O-glucuronidation two types of glucuronidation happen one with ethers and other with

esters. Aliphatic alcohol and phenols follows O-ether glucuronidation while carboxylic acids

follows O-esters glucuronidation(Chen, LeDuc et al. 2010).

O-ether glucuronidation metabolizing drugs contain Alcohol and phenol: buprenorphine,

codeine, lorazepam, propofol, the zidovudine, propranolol, salicylic acid etc.(Stachulski and

Jenkins 1998).

O-esters glucuronidation metabolizing drugs contain Carboxylic acid: diclofenac, ibuprofen,

mycophenolic acid, valproic acid, salicylic acid etc.(Tsukamoto, Kato et al. 1964).

Similarly,S-glucuronidationmetabolizing drugs contain Sulfhydryl and thiol functional

group:disulfiram, methimazole, propylthiouracil, diethyl thiocarbamic acid etc. (Testa 2007).

1.1 GLUCURONIDE ENZYMES

Enzymes are the biological catalysts involved to accelerate the chemical reactions. In

glucuronidation metabolism the enzyme “uridine diphosphate glucuronosyltransferase

(UGT)” is involved in which uridine diphosphate glucuronic acid act as co-factor and it

contains a carboxylic group which get ionized at physiological pH and finally it produces a

compound with low biological activity and high-water solubility and are more ready for

excretion (Guillemette 2003). In the clearance of exogenous and endogenous substances,

glucuronidation is the rate-limitingstep. Glucuronide enzymes are membrane-bound enzymes

and mainly excreted through renal and biliary systems (Jancova, Anzenbacher et al. 2010).In

general uridine diphosphoglucuronate enzyme transfer the glucuronyl group to the

compounds having nucleophilic functional groups such as nitrogen, carbon, oxygen or

sulphur (Bruggeman and Darras 2009).


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By using a sugar co-factor i.e. UDPGIcUA, enzyme glucuronyl transferase transfer

glucuronic acid (substrates) to form β-glucuronide and β-D glucopyranosiduronic acid(Ritter

2000). The enzyme UDP-glucuronyl transferase also plays an important role in the drug

development phase because the majority of the drugs undergoes metabolism through this

enzyme (Oda, Fukami et al. 2015). Polymorphism in UGT enzymes plays a vital role in the

metabolism of drugs. It can change the pharmacokinetic parameters.Alteration to these

parameters are important in manifestation of the genetic defect. These changes in different

populations are observed and in some cases drugs or allelic combinations shows a weaker

genetic effect (Stingl, Bartels et al. 2014). Due to this polymorphism on UGT enzymes as

genetic imminence causes a large variety of cancers in bladder, breast, colorectal,

endometrial, oesophageal, head, neck, lung, liver, prostate and thyroid. The reason behind

developing cancer is reduced UGT enzymatic activity or expression. UGT activity plays an

important role in the metabolism and clearance of carcinogens. Sometimes this

polymorphism may not directly cause cancer, but it is associated with cancer causative

polymorphs like UGTor non-UGT genes closely linked carcinogenicgenes. In the humanbody

UGT enzymes are involved in apoptosis, cellular metabolism, oxidative stress and

carcinogenesis. Involvement of nuclear UGTs causes the retinoid, steroids and possible fatty

acids biological activity termination (Fujiwara, Yokoi et al. 2016).

1.2 Isoforms of UGT

Based on the sequence of amino acid 22 UDP genes are identified and these are further

classified as families and subfamilies. These enzymes are comprised of four families like

UGT1, UGT2, UGT3and UGT8.

As we know UGT1A1 plays an important role in bilirubin metabolism, if the mutations occur

in UGT1A1 it will cause the congenital, non-haemolytic, unconjugated hyperbilirubinemia

known as Gilbert’s syndrome and Crigler-Najjar (Shiu, Huang et al. 2015). Several studies

suggested that mutations in UGT1A1 can increase the risk of neutropenia and diarrhoea(Li,

Wang et al. 2014). UGT1A3 involved in glucuronidation of Atorvastatin, a polymorphism on

these enzymes closely related to UGT1A1 and finally it will effect on the pharmacokinetic

and lipid-lowering effect of atorvastatin (Cho, Oh et al. 2012). Single nucleotide

polymorphism (SNP) is observed in human UGT1A3. The activity of quercetin, luteolin,

kaempferol can be altered by UGT1A3 variant’s (Chen, Chen et al. 2006). UGT1A3 is

responsible for the formation of nor-UDCA 23-G (Nor-ursodeoxycholic acid 23-glucuronide)


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in humans (Trottier, El Husseini et al. 2010). UGT1A4 involved in the N-glucuronidation of

the lamotrigine and excreted as 2N or 5N glucuronide conjugates through urine(Gulcebi,

Ozkaynakcı et al. 2011). During pregnancy, the response of a patient with respect to

medication will be different. In these condition the clearance level also vary, some of CYP

enzymes like CYP3A4, CYP2D6 and CYP2C9 and UGT mainly UGT1A4 increases the

clearance of the drugs that undergo metabolism through these enzymes(Pennell, Newport et

al. 2004). UGT1A6 is one of the important co-former in UGT family, generally, a

polymorphism on these enzyme doesn’t effect on serum valproate concentration(Chu, Zhang

et al. 2012).UGT1A6 is useful for the carcinogen’s detoxification like BaP (benzo[a]pyrene)

in cigarettes. UGT1A6 catalyse glucuronidation of planar and small molecules like phenol,

indole and coumarins(Ghemtio, Soikkeli et al. 2014). Polymorphism on UGT1A6 is a risk

factor for developing breast cancer because it is responsible for steroid hormones

conjugation too, so it will increase the level of steroid hormones (Lampe, Bigler et al. 1999).

HNF (hepatic nuclear factors) are of different isoforms, in this HNF1 alpha and HNF4 alpha

regulates the expression of UGT1A7. HNF1 alpha is high in activating the expression of the

enzyme as compared to HNF4 alpha (Ehmer, Kalthoff et al. 2010). UGT1A8 primarily

catalyse the DHT (dihydrotestosterone), it catalyse DHT diglucuronides form to DHT

monoglucuronides form as well(Murai, Samata et al. 2006). UGT1A9 is the primary

metabolic enzyme for the glucuronidation of the mycophenolic acid and propofol, it alter the

pharmacokinetics of these drugs whenever it undergoes single nucleotide polymorphism

(Wang, Zhang et al. 2017). UGT1A10 mainly contribute in presystemic first pass metabolism

because it is mainly expressed in the intestine. In general,UGT1A10 is absent in liver due to

hypermethylation of UGT1A10 promoters at CpG-rich region(Oda, Fukami et al. 2014). For

the metabolism of polycyclic aromatic hydrocarbons UGT2A1 are the main responsible

detoxification enzyme, due to polymorphism on UGT2A1 it increase the risk of tobacco-

related cancers(Bushey, Chen et al. 2011). UGT2A2 is a functional enzyme and mainly

expressed in nasal mucosa(Sneitz, Court et al. 2009). UGT2A3 contain two common variants

like (UGT2A3.1 and UGT2A3.2) and enzyme affinity constant differ for both the variants.

Pregnancy X-factor (PXR. NR112) ligand induces the expression of UGT2A3 in human

intestinal cell lines (Hazarika, Krishnaswamy et al. 2008). UGT2B7 is a direct target for the

mi-R3664-3p in liver cancer cells (Wijayakumara, Mackenzie et al. 2017). Whatever drugs

use UGT2B7 glucuronidation as a primary elimination, aprepitant may alter the clearance of

these drugs because it is the inhibitor of the UGT2B7 (House, Ramirez et al. 2015).

UGT2B10 as an orphan enzyme as it consist of less amino acid sequence, for nicotine hepatic


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N-glucuronidation UGT2B10 is the main contributing enzyme (Kaivosaari, Toivonen et al.

2007). A diversified transcriptome of UGT2B10 was created by alternative splicing and it

represents UGT2B10 half expression in human liver. Amine-containing drugs like

antipsychotics and tobacco metabolites undergo UGT2B10 dependent detoxification (Fowler,

Kletzl et al. 2015). UGT2B11 interact with some of other UGT enzyme to produce a

differential enzymatic complex at the same time it functions similar to UGT2B10(Beaulieu,

Lévesque et al. 1998). Modulations of UGT2B15 and UGT2B17 levels are differential during

prostate cancer progression, in these UGT2B15 levels are decreased and UGT2B17 levels are

increased or unchanged(Paquet, Fazli et al. 2012).

1.3 Mechanism of glucuronidation

Glucuronidation reaction is catalysed by membrane-bound UGTs; mainly these enzymes are

present in the endoplasmic reticulum. The compounds like endogenous and exogenous

toxins, polycyclic and simple phenols in presence of UDPGIcUA enzyme undergoes

glucuronidation and form β-D glucopyranosiduronic acid or glucuronide derivatives and the

resulting glucuronides contain D-glucopyranuronosyl radicals which linked to form O-

glucuronides ethers, O-glucuronide esters, N-glucuronides, S-glucuronides and C-

glucuronides.

1.4 Influencing factors for glucuronidation metabolism

Administration of foreign compounds and hormones and by the age, sex, strain and some of

the diseases can affect on drug metabolising enzymes in the liver.

2. INVITRO STUDIES FOR GLUCURONIDATION

In-vitro studies are important in drug discovery process, mainly useful for metabolic stability,

metabolite generation for bioanalytical assay development and also find out species

differences in metabolism (Fisher, Campanale et al. 2000).

Methods for In-vitro metabolism studies

i. Microsomal glucuronidation assay

ii. Hepatocytes in-vitro method

iii. Recombinant enzymes method

iv. Subcellular fraction method

v. Liver slices method


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Coming to the glucuronidation more common microsomes and hepatocytes in-vitro methods.

i. MICROSOMAL GLUCURONIDATION ASSAY

Microsomes (400µl-aliquots) were activated by using 4 x 5-s bursts sonication (microson

ultrasonic cell disruptor) allowing 1min on ice between bursts. After the microsomal

activation, prepare a combined volume of 100μl it should contain 100 mM tris or maleate

buffer, pH 7.4 and in these buffers add substrate, 10mM saccharic acid and 10mM MgCl2.

After the preparation of combined volume, keep it into an incubator at 37°C for 60min and

prechilled to -20°C before the prechilling add 100μl methanol for termination. After that

transferred to a centrifuge and the mixture was centrifuged for 10 min at 14,000g and finally,

the resulting supernatant was analysed by using HPLC(Soars, Burchell et al. 2002).

ii Hepatocytes in-vitro method

Here mainly two steps are involve

a) preparation of freshly isolated rat hepatocytes

b) In-vitro metabolism in rats

a. Preparation of freshly isolated rat hepatocytes

By using in-situ collagenase method prepare the rat hepatocytes. These are purged with 95%

O2, 5% CO2, after that suspend in William’s medium E containing L-glutamine. Trypan blue

exclusion test is used for the assessment of cell viability.

b. In-vitro metabolism in hepatocytes

Drug compound is incubated with hepatic suspension reaction mixture. Following

preincubation for 5min at 37°C reaction mixture started by adding 5µl of drug compound

solution in methanol and final concentration used is 1µM.The reaction stopped by adding

chloroform, ethyl acetate and acetonitrile after incubating at 37°C for different time interval.

After termination, centrifuged for 5min at 10000g, supernatant will be collected and organic

fraction evaporated after adding internal standard reconstituted the residue with mobile phase

and analysed by HPLC (Naritomi, Terashita et al. 2003).

3. Characterisation of glucuronide Metabolites

There are fast development in recent analytical methods like HPLC, UPLC, GC, CE, coupled

to NMR and MS helps in fast separation, detection, quantification and characterisation of

metabolites. LC coupled to MS provides useful information about the metabolic reaction and


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about parent drug atom where metabolism occurs. There are three major tasks in drug

metabolism firstly to elucidate the structural moiety of parent compound which cause inferior

exposure and high metabolic clearance. Secondly to identify the functional group prone to

undergo metabolism to form reactive and toxic metabolite and lastly to explain the PK/PD by

observing and monitoring the therapeutically active metabolite. The various detectors used

for identification include UV (ultraviolet), MS (mass spectrometry) NMR (Nuclear magnetic

resonance) analyser and radioactivity detectors(Nnane and Tao 2005).

Some examples of metabolite identification and their characterization is discussed in the

following section:

Osthenol is used as anti-inflammatory, anti-bacterial, anti-tumour and anti-fungal. The major

metabolic pathway of osthenol is glucuronidation was developed on LC-MS/MS method.

Chromatographic conditions for separation of metabolites were using Phenomenex Kinetex®

C18 column having dimension of 150 mm x 2.1 mm, 2.6 µm and mobile phase used was

(solvent A) water and (solvent B) Acetonitrile both containing 0.1% formic acid in gradient

mode, it follows as 0-21 min 15% - 57% of B is used followed by 21-12min 57%-95% of B,

then for 23-24min 5% of B, at 24-25.1min 95%-15% of B and then for 25.1-30min 15% B

been used at a flow rate of 0.2 ml/min at 45⁰C. and mass spectrometry was performed in

negative ionisation mode. Osthenol O-glucuronide conjugate observed at 7.0 min with m/z of

405 and hydroxyl-glucuronide shows m/z of 421 at 8.0min (Cho, Paudel et al. 2018).

Similarly, Picroside II shows antioxidant, anticholestatic, hepatoprotective and immune

modulating pharmacological activities. An LC-MS and HPLC methods were developed on

Hypersil ODS2 C18 analytical column with dimensions 150mm x 4.6 mm, 5 µm; these

column been protected by Phenomenex guard column using mixture of 0.03% ammonium

acetate v/v ( solvent A) and methyl alcohol (Solvent B) as mobile phase set in gradient flow

as 80:20 v/v (A:B) to 65:35 v/v (A:B) for 0-5min, 65:35 v/v (A:B) to 80:20 v/v (A:B) for 8-

12min and then 80:20 v/v (A:B) for 4 min with flow rate of 1.0 ml/min. The autosampler

temperature and column temperature was maintained at 4⁰C & 40⁰C respectively. Mass

spectrometry operated in selected ion monitoring mode and Picroside II glucuronide obtain in

negative mode. Data processing is done on version 3.20 Shimadzu LC-MS solution software.

In HPLC the column is same with dimensions 250mm x 4.6mm, 5µm. The mobile phase used

is mixture of water and methanol containing 0.04% Triethylamine (52:48 v/v) pH 4.5

adjusted with formic acid. Column temperature and flow rate maintained at 40ºC and 0.8

ml/min respectively. SPD 10A UV detector is used and scanned at 260nm. Data acquisition


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and analysis were done by using HS Chromatography Data System software. The

Glucuronide metabolite formation confirms by treating with β glucuronidase hydrolysis assay

where there is no change in control sample while after incubating with β glucuronidase the

picroside II glucuronide decrease (Li, Zheng et al. 2011). An endogenous substance like

bilirubin which is the main component of bile pigment its metabolites quantified by using

reverse phase DiamonsilTM

C18 column with dimensions 20mm x 4.6mm, 5µm, protected

with Inertsil ODS-SP cartridge guard column having dimensions 10mm x 4mm. The mobile

phase used is formic acid (0.1%) containing (Solvent A) water and (solvent B) acetonitrile in

gradient mode as follows 40%-75% of B for 0-9 min, 75%-95% of B for 9-18 min, 95% of B

for 18-27min, 95%-40% of B for 27-30 min with flow of 1ml/min. The column temperature

set was 45ºC. UV-Vis detector used and scanned at 450nm. Injection volume was 100 µl.

Data collection and analysis were done by using Shimadzu LC solution workstation software.

Bilirubin metabolites that are BMG1 and BMG2 (bilirubin monoglucuronide metabolite and

isomers) were observed as BMG1 IX-α and XII-α at 8.564 and8.940 min respectively.

Similarly, BMG2 identified as BMG2 III-α and IX-α at7.571 and 7.950 min likewise BDG

metabolite (bilirubin diglucuronide metabolite) identified as BDG III-α, IX-α, and XIII-α at

4.515,4.914 and 5.346 min respectively (Ma, Lin et al. 2014). Moreover, the new multiple

kinase inhibitor is employed for the cancer therapy like Regorafeniband its metabolites

separated by UPLC-Q-TOFMS using Acetonitrile containing formic acid (0.1%) in gradient

flow for 16 min at flow rate of 0.3 ml/min. Metabolites separated using XDB-C18 column

having dimension 2.1×100mm,1.8µm.Column temperature set at 45⁰C. Positive ionisation

mode set in mass spectrometry. Data acquisition obtain by Agilent mass hunter workstation

Software. Mass Profinder version 6.00 used for manipulating the collected raw mass

data,OPLS-DA (Orthogonal Partial Least-Squares Discrimination Analysis) done by

SIMCAP+13.0 software. Regorafenib metabolites i.e. M10 and M11 detected at m/z of

659.1146 and 675.1125 at 8.40 and 7.50 min respectively(Wang, Xiao et al. 2018).

Wedelolactone (WEL) shows a wide range of pharmacological activities like anti-

inflammatory,antiosteoporosis,anticancer, anti-adipogenesis, anti-hepatitis C virus, anti-

hepatic fibrosis, trypsin inhibition, angina pectoris, venom poisoning etc. Wedelolactone

undergo mainly methylation and glucuronidation. The functional group 5-OH is mainly prone

to glucuronidation The metabolites are seperated using front end as Acquity UPLC HSS T3

column with column dimensions 100mm x 2.1mm, 1.8µm and mobile phase consist of

mixture of ammonium acetate (5mM) (solvent A) and methanol (solvent B), run with


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gradient : 5% of B to 70% of B for 15 min and then run at isocratic mode for 1 min with

70% of B and again increased for 1min upto 95% of B , and isocratic for 3min with 95% of B

and at last down to 5% of B. The column temperature maintained at 40ᴼc, flow rate

maintained at 0.4ml/min. UV detection is done at a wavelength of 351nm, mass spectrometry

performed in positive ionisation mode. Information Dependent Acquisition (IDA) method

performed by using software (Analyst 1.6) (Li, Huang et al. 2016). WEL glucuronide

metabolites M5-1, M5-2 and M5-3 eluted at 7.2min, 8.6min, and 9.3min respectively with

m/z of 491.0852. Similarly, M6-1, M6-2, M6-3, M6-4 are eluted at 8.1min, 8.8min, 8.9min

and 10.0min at m/z of 505.098. Metabolite M8-1 and M8-2 eluted at 3.9min and 6.1min with

m/z of 681.130. M8-1 metabolite product ion spectra not detected due to less concentration in

the sample. The diglucuronide metabolites M7-1 and M7-2 obtained at m/z of 667.1147 and

667.1120 which is eluted at 3.8min and 5.3min respectively (Li, Huang et al. 2016).

Icaritin possess several biological and pharmacological activities like immunomodulatory and

neuroprotective effects, protection against bone metabolism and also prevent osteonecrosis

associated with steroid icaritin also used in anticancer therapy.Metabolites were quantified

using formic acid (0.1% v/v) in water (A) and formic acid (0.1%) in acetonitrile with

gradient flow of 90% A for 0-0.5min,90-60% A for 0.5-1min, 60%A for 1-1.5min, 60-40%A

for 1.5-2min,40-5%A for 2-2.5min,5-90%A for 2.5-4min, with flow rate of 0.5ml/min.

Metabolite separated on Waters BEH C18 column with column dimensions 2.1 mm x 50 mm,

1.7μM. Column temperature set at 40ᴼC. Mass spectrometry performed in positive ionization

mode. Icaritin metabolites observed at 1.96min icaritin-3-glucuronide (G1), icaritin-7-

glucuronide (G2) at 2.06min and 1.36minas icaritin-3,7-diglucuronide (G3) with m/z of

545.35,545.35 and 721.34 respectively (Rong, Meng et al. 2018).

Methyl Gallate (MG) and Pentagalloyl Glucopyranose (PGG) shows various pharmacological

activities like antibacterial, anti-inflammatory, antiviral, anti-enzymatic, anticancer and anti-

oxidant.MG AND PGG metabolites separated by using KinetexC18 column with dimensions

50mm × 2.1mm, 5µm. The mobile phase consist of formic acid (0.1%) as solvent A in

deionized water and acetonitrile (100%) solvent B run in gradient flow as 0% B for 0.0-

0.5min; 0-30% of B for 0.5-1min; 30-90% of B for 1.0-2.0min; 90% of B for 2.0-3.0min; 90-

0% of B for 3.0-3.5min; 0% of B for 3.5-5.0min at a flow rate of 0.5ml/min. The sample and

column temperature set at 20ᴼC & 40ᴼC. 10µl was the injection volume. Mass spectrometry

operated in negative ionization mode. MG metabolites identified as M1 and M2 MG


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glucuronide with pseudo-molecularions of m/z 359.1 (M1) and 358.9 (M2) at 1.8 and 1.9min

and PGG metabolite i.e. PGG glucuronide observedwith pseudo-molecular ions of m/z

1115.1 at 1.9min (Jiamboonsri, Pithayanukul et al. 2016).

Ursolic acid possess various pharmacological activities like anti-tumour, anti-inflammatory,

antioxidative, antiherbal activities. Chromatographic method was developedon LC-MS/MS

method to separate the metabolites using mobile phases consist of 5mM of ammonium

formate (pH 3.41) and acetonitrile (20:80 v/v) with a flow rate of 0.2ml/min. The column

used is Luna C18 with dimensions 50mm x 2.0mm, 5µm; injection volume was 5µl.Mass

spectrometry performed in both positive and negative ionisation mode.Ursolic acid

metabolite observed at 2.8min with m/z of 631.30 in negative ionisation mode. Hydrolysis

with β glucuronidase also confirms the presence of glucuronide metabolite since with time

there is an increase in ursolic acid (Gao, Liu et al. 2016).

Trovafloxacin has an antibiotic activity. Metabolites were quantified using Mightysil RP-18

GP column with dimensions 4.6mm x 150mm, 5µm. The mobile phase consists of 20mM of

phosphoric acid containing 18% of acetonitrile with a flow rate of 1.0ml/min. metabolite was

detected using a fluorescence detector with 450nm emission and 280nm excitation

wavelength. Theformation of glucuronide metabolite confirms by reacting with β-

glucuronidase enzyme and compare the peak area. It was found that with time there is a

decrease in the peak area of trovafloxacin glucuronide. The retention time of trovafloxacin is

2.4min (Fujiwara, Sumida et al. 2015).

Curcumin possess a wide range of pharmacological activities like antioxidant, flavouring and

colouringagent,anti-inflammatory, anticancer etc., metabolites were separated using

Phenomenex RP Luna C18 column with dimensions, 250mm x 4.6mm, 5µm; mobile phase

consisting of deionized water (solvent A) maintained at pH 3.0 with formic acid and

acetonitrile (solvent B). Separation achieved using linear gradient run, for curcuminoids, 30-

70% of B for 0-35 min were runand for hexahydro-curcuminoids15% of B for 0-6min, 15-

30% of B for 6-7min, 30-35% of B for 7-17min and 35-45% of B for 17-32min. Flow rate

setting was 1.0ml/min. Data collection and analysis were done by HP Chemstation software.

The same mobile phase used as HPLC in LC-MS except for a Phenomenex synergy hydro

column with dimensions 250 x 4.6mm, 4µm is used. Detection was done by Diode array

detector, curcuminoids and hexahydro-curcuminoids measured at 420nm and 280nm

respectively. Mass spectrometry operated in negative ionisation mode. Curcumin glucuronide


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confirms by reacting with β glucuronidase enzyme.Curcumin glucuronide, hexahydro-

curcumin glucuronide,observed at m/z of 543,549 respectively (Hoehle, Pfeiffer et al. 2007).

Hesperetin and Ferulic acidmetabolites identified by developing HPLC-MS/MS method.

Phenomenex kinetex XB C18 column with dimensions 2.1mm x 100mm, 2.6µm; column

temperature maintained at 35ºC. The mobile phase consists of Milli-Q containing 5% of

Acetonitrile and 0.1% of Formic acid (solvent A) and Acetonitrile containing 5% of Milli-Q

and 0.1% of formic acid (solvent B). Hesperetin metabolite separated using gradient flow like

12% of Bisocratic for 0-22min, 12-35% of B for 22-32min, 35-90% of B for 32-33min, for

33-35min 90% of B on isocratic mode, 90-12% of B for 35-36min and 12% of B on isocratic

for 36-50min. For ferulic acid gradient flow set as 5% of B isocratic for 0-1min, 5-35% of B

for 1-10min, 35-90% of B for 10-11min, 90% of B isocratic for 11-13min, 90-5% of B for

13-14min and 5% of B isocratic for 14-30min. Flow rate setting was 0.25ml/min and

injection volume kept was 10µl. Mass spectrometry performed in negative ionisation mode.

Hesperetin glucuronide metabolites found at m/z of 477 while ferulic acid metabolites

identified at m/z of 369 respectively(Van Rymenant, Abranko et al. 2017).

Tolvaptan is used in the treatment of hypervolemic, hyponatraemia and euvolemic and also in

congestive heart failure and cirrhosis. LC-MS(/MS) method develop to separate the

metabolites of tolvaptanwhere LC system equipped with API4000 ABSciex triple quadrupole

and Agilent Technologies 6520 orthogonal acceleration time-of-flight mass spectrometer.

Triple quadrupole and time of flight both operated in positive and negative ionisation mode

but full scan spectrum in negative mode no signal obtained and in positive mode abundant

signal was recorded for M+H protonated molecular ion. In triple quadrupole Analyst

Software Version1.6.1 were used to manage the instrument control, sample injection, method

set up parameters and operating the sequence. In a time of flight Agilent Technologies Mass

Hunter software version B.02.01 were used to manage the instrument control, sample

injection, tuning, method set up a parameter and operating the sequence. Metabolites were

separated using reverse phase column with dimensions 10cm column length and 2.1mm

internal diameter and 2.7μm particle size. The column temperature set at 40°C. The mobile

phase consisted of Acetonitrile (solvent A), water (solvent B) and 0.1% formic acid is used as

mobile phase modifier run in the gradient mode as 10% of B for 10min, 30% of B after 4

min, after 3min 40% of B,after 5min 60% of B and 100% of B then after 4min at flow rate of

0.25ml/min. 10µl was the injection volume. The M6, M11 and M15 metabolites of tolvaptan


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undergoes phase II after phase I. These metabolites eluted at 9.7min, 11.3min and 11.8min

respectively(Mazzarino, Buccilli et al. 2017).

Bavachinin (BCI) possess several pharmacological activities like in treatment of asthma

osteoporosis, vitiligoand another different skin disease. BCI also shows anti-inflammatory,

analgesic, antipyreticactivities. Metabolites of BCI separated using LC-MS system with

Shim-pack XR-ODS column with dimensions internal diameter 2.0mm x 75.5mm, 2.2μm,

protected with ODS guard column internal diameter 2.0mm x 5mm, 2.2μm. Column

temperature maintained at 40°C. BCI and its metabolite scanned at 275nm using SPD-M 20A

diode array detector. The mobile phases used is (Solvent A) CH3CN and (solvent B)

water/0.2% formic acid, run at gradient mode as 90-75% of B for 0-1min, 75-48% of B for 1-

8min, 10% of B for 8-11min, and again 90% of B for 11-15min, at a flow rate of 0.4ml/min.

Mass spectrometry operated in positive mode. Data acquisition is done by using LC-MS

solution software version 3.41. The major metabolic pathway of BCI is glucuronidation, the

BCI glucuronide found at m/z of 515 (Lv, Hou et al. 2015).

F18 (10-chloromethyl-11-dimethyl-12-oxo-calanolide) Ais a nonnucleosidereverse

transcriptase inhibitor (NNRTI) used in the treatment of HIV. M3 is the most commonly

formed metabolite of F18.HPLC-MS/MS method were developed to separate the metabolites

using Zorbax SB-C18 column with dimensions 3.5mm, 2.1mm x 100mm; mobile phase

consists of water (solvent A) and Acetonitrile (Solvent B). Gradient program set as 5% of B

for 0-1.5min, 90% of B for 1.5-1.6min, 90% off for 1.6-7.0min, 5 %of B for 7.0-7.1min and

again 5% of B for 7.1-9.0min. Flow rate setting was 200µl/min with the operating

temperature kept on 25°C, 5µl was the injection volume. Mass spectrometry operated in

positive ionisation mode. Data collection and reporting did by using Xcalibur 1.4 software.

The formation of M3-O-glucuronide confirms by reacting with a β-glucuronidase enzyme

(Liu, Sheng et al. 2015).Homoegonolpossess various pharmacological activities like

antihistaminic, anti-inflammatory,anti-asthmatic and antimicrobial. Metabolites quantified by

developing LC-QTOF method to identify the metabolite of homogenous and LC-MS/MS

(tandem mass spectrometry) to identify the UGT and CYP isoform responsible for the

metabolism of homogonous.Homoegonol and its metabolite separated using mobile phase

methanol (5%) in formic acid (0.1%) (Solvent A) and methanol (95%) in formic acid (0.1%)

(solvent B) run at gradient mode as for 0.5min 40% of B, for 0.1min 40-95% of B, for 4.9min

95% of B, for 0.1min 95-40%of B, for 4.4min 40% of B with 0.3ml/min of flow rate. The


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temperature of autosampler and column were maintained at 6˚C and 50˚C respectively.

Column used is pinnacle biphenyl column with dimensions of (i.d.) 2.0mm x 100mm, 5µm.

QTOF mass spectrometry operated in positive ionisation mode. Data acquisition and analysis

were done by using Mass Hunter software. In tandem quadrupole mass spectrometry

Xcalibur® software used to integrate the peak areas. Injection volume was 5µl. Glucuronide

metabolites M4 and M5 are seen at m/z of 505.16967 and519.18554 respectively(Kwon, Kim

et al. 2015).

Bisphenol S(BPS) is used to produce thermal paper and polycarbonate plastic as an

alternative to Bisphenol A. Metabolites were Separated using pro shell C18 column with

dimensions 100mm x 3.00mm, 2.7µm.Mobile phaseconsists of ammonium acetate (10mM)

in milli-Q water (solvent A) and Acetonitrile (solvent B) run in linear gradient mode as

follows 3% B for 0-3min, 3%-90% of B for 3-10min, 90% of B for 10-15min, 90%-3% B for

15-16min and then re-equilibrate the column till 20min. Column temperature set was 50ᴼC.

injection volume was 1µl. Mass spectrometry operated in negative ionization mode. BPS

glucuronide eluted at 5.58in with m/z of 425.1 and BPSM1 with m/z of 441 eluted at 6.6min

(Skledar, Schmidt et al. 2016).

Fraxetin possess anti-atherosclerosis effects, it been used in human neuroblastoma cells and

act as neuroprotective agent. Fraxetin and its metabolites separated developing a method

using chrome neutral amide analytical column with dimensions 150mm x 4.6mm, 5µm. The

mobile phase used is acetonitrile (solvent A) and 0.2% formic acid in water (solvent B) run at

a gradient of 98%-78% of B for 0.0-10.0min, 10% of B for 10.0-18.0min and 98% of B for

18.0-28.0min at a flow rate of 1ml/min. The column temperature setwas 40ᴼC. The detector

used is SPD-10AVP UV at a wavelength of 338nm. Mass spectrometry operated in negative

and positive ionization mode but negative ionization was selected because it is more sensitive

than positive ionization mode.Data collection and analysis were done by using LC/MS

Solution software version 3.41. NMR spectrometry performed on INOVA-400 NMR

spectrometer using DMSO-d6 as a solvent. M-1 and M-2 metabolites were observed at

8.5min and 9.5min with m/z of 383 indicating the mono-glucuronides metabolites of fraxetin.

This metabolite formed also confirm by reacting with a β-glucuronidase enzyme (Xia, Liang

et al. 2014).

Opioid drug Morphine used to cure the pain associated with severe acute to chronic cancer

pain. Metabolitesof morphine separated using mobile phase potassium phosphate (0.01M)


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maintain at pH 2.2 and acetonitrile (85:15 v/v) having sodium dodecyl sulfate (0.04mM). The

column used is Spherisorb®

ODS2 reversed-phase column with dimensions 250 x 4.6 x 5μm.

Extraction technique used in Solid Phase Extraction.Detection was done using DAD at

210nm. Flow rate maintained was 1ml/min. injection volume was 40µl. Waters Millennium32

software used for chromatogram analysis. Optimal runtime for analysis is 40min. The

metabolites morphine-3-glucuronide and morphine-6-glucuronide observed at 9.8min and

15.1min respectively(Oliveira, Carvalho et al. 2014).

Calycosin is an isoflavonoid having Qi-tonifying effects. Metabolites were separatedby using

HPLC-DAD or HPLC-MS/MS method. In HPLC- MS/MS method develop using reverse

phase Agilent ODS C18 column with dimensions 250mm x 4.6mm, 5µm; mobile phase used

is 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) in gradient mode as 5%-

39% of B for 0-33.0min,33%-100% of B for 33.0-40.0min. Mass spectrometry operated in

negative ionization mode. HPLC- DAD performed using the same column and gradient

mobile phases as that of HPLC-MS/MS and protected with C18 ODS reverse phase guard

columnhaving dimensions 12.5mm x 4.6mm, 5µm. The column temperature set was 25ᴼC

and flow rate maintain was 1ml/min. Metabolites scanned usingDAD detector at 250nm.70µl

was the injection volume.NMR spectrometry performed using Bruker AV-600 NMR

spectrometer using DMSO-d6 as a solvent. The metabolites G1 and G2 eluted at 19.3min and

22.8min respectively. The quasi-molecular ion peak of both metabolites shows at m/z of 459

indicating conjugation with glucuronic acid (Ruan and Yan 2014).

Resveratrol and Pterostilbeneare commonly found in grapes and berries, Pterostilbene is

dimethylated analog of resveratrol. Resveratrol possess many pharmacological activities like

anti-oxidant, antiaging, anti-inflammatory and anticarcinogenic. Pterostilbene is more potent

than resveratrol in skin, liver and colon cancer. Metabolites were separated by developing a

method using HPLC Atlantis T3 C18 column having dimensions of 6mm x 250mm, 5µm.

The mobile phase used is (solvent A) 20mm ammonium acetate pH 5.0 and (solvent B)

methanol with gradient elution as 90% of A for 4min, then for 20min 80% of B, 95% of B for

4min and re-equilibrate for 2min with initial conditions. Flow rate maintained was 1ml/min.

Detection of metabolites done by using UV detector at 308nm. Resveratrol

metabolitesresveratrol-3-O-glucuronide and resveratrol-4-O-glucuronide observed at 14 and

13min respectively. Similarly, Pterostilbene metabolite pterostilbene-4-O-glucuronide

observed at 20min. Further,these metabolites confirm by reacting with β-glucuronidase


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enzyme and they observed only the resveratrol and Pterostilbene standard peak (Dellinger,

Garcia et al. 2014).

20 (S) protopanaxatriol (PPT) possess cardioprotective activities in myocardial ischemic

injury. Metabolites quantified using (solvent A) water and (solvent B) acetonitrile in gradient

mode as 2%-8% of B for 0-2.0min, 8%-18% of B for 5-9min, 28%-60% of B for 9-14min,

60%-90% of B for 14-19min and then re-equilibrate for 4min with 2%of B. Column used is

ACQUITY UPLC HSS T3 column with dimension 100mm x 2.1mm,1.8 µm; column

temperature maintained at 45ᴼC. the flow rate was 0.4ml/min. Mass spectrometry operated in

negative ionisation mode. Raw data acquisition and analysis done by MassLynxTM

Version4.1 software,MetaboLynxTM

softwareis used for post-acquisition and data analysis.

NMR spectrometry performed on Bruker AV 400 NMR spectrometer using C5D5N solvent at

25ᴼC. Glucuronide metabolites of PPT M20, M21 and M23 observed at11.63min, 11.75min,

and 13.51min respectively with m/z of 651.4102 (He, Zhou et al. 2014).

Fosinopril is an ACE (angiotensin-converting enzyme) inhibitor and used in the treatment of

hypertension. Fosinopril is a prodrug it forms active metabolite fosinoprilat. Metabolites

separated using Zorbax Eclipse plus C18 rapid resolution HD column with dimensions 2.1mm

x 50mm,1.8µm. in front of column inner filter of 0.2µm is used. Mobile phase used is

(solvent A) 10mM aqueous ammonium acetate maintain at pH 5.4 and (Solvent B) is

acetonitrile in gradient elution as for 0.5min 5% of B, 0.5-15min 5%-50% of B, 15-15.1min

50%- 90% of B,15.1-17.5min 90% of B,17.5-17.6min 90%-5% of B,17.6- 20min 5% of B is

used with flow rate of 0.6ml/min. the temperature of the sample tray was kept at 8ᴼC.For

0.5min the flow was into the waste to restrict the inorganic ions moving in the mass

spectrometer. For partial loop with needle overfill, 5µl injection volume was used and 3µl

volume is used for overfill flush. Mass spectrometry operated in both ionization mode i.e.

positive and negative. In positive ionization mode glucuronide metabolites M44 and M45

characterized at m/z of 634 and 762 with sodium adduct formation and in negative ionization

mode, M44 and M45 observed at m/z of 610 and 738 respectively (Uutela, Monto et al.

2014).

Glycycoumarin (GCM)shows various biological activities like antispasmodic, anti-

inflammatory, anti-oxidant, anti-hepatitis C virus and also inhibit smooth muscle contraction

due to stimulants like KCl and BaCl2. Method developedon HPLC-MS and HPLC-IT-TOF-

MS method to separate the GCM metabolites. HPLC/DAD/ESI-MS develop using agilent


1508


zorbax SB-C18 column having dimensions 4.6mm x 250mm, 5µm. Column temperature

maintained was 30ºC. The analytical column protected with Zorbax Extend- C18 guard

column with dimensions 4.6mm x 12.5mm, 5µm. Mobile phase consisted of acetonitrile

(solvent A) and 0.1% formic acid in water (solvent B) eluted in linear gradient as 12% of A

for 0min, 39% of A for 30min, 44% of A for 40min, 95% of A for 50min, 95% of A for

55min at a flow rate of 1.0ml/min. DAD detector used and spectra are obtained by scanning

at 200-400nm.Mass spectrometry performed in negative ionisation mode and data acquisition

and analysis done by Xcalibur2.0.7 software. In HPLC-IT-TOF-MS analysis the HPLC

conditions were similar to that previously described. Data collection and analysis were done

by using LC-MS solution software. The glucuronide metabolites i.e. M6, M10, M12 observed

with m/z of 543 and identified as GCM glucuronides whereas M1 and M2 are identified as

hydroxyl GCM glucuronides with m/z of 559(Wang, Qiao et al. 2014).Steviol is the

hydrolysis product of stevioside A. Metabolites separated using An AgelaVenusil XBP

C18column with dimensions 50 x 2.1 mm;5µm. Mobile phase consists of (solvent A) formic

acid (0.1%) and (solvent B) formic acid (0.1%) in methanol at a flow rate of 0.3ml/min. The

column temperature was 40ᴼC. Mass spectrometry performed in negative ionization mode.

The glucuronide metabolites undergo a transition from 493.2 to 317.3 m/z with an intense

and characteristic peak confirms the glucuronide metabolite. Data collection and analysis

were done by using AB Sciex Analyst 1.5.2 software (Wang, Lu et al. 2014).Silybin is

flavonolignan found in silymarin complex.Silybin is used as anticancer, antioxidant,

hepatoprotective, chemoprotective, chemoprotective and also possess hypocholesterolemic

activities. Method developed to separates the metabolites of silybin using water (solvent A)

and acetonitrile (solvent B) containing 0.1% formic acid mobile phase in gradient mode as

100%A for 0-1.5min, 0-100%B for 1.5-15min, and 100-0%B for 16-16.5min. Column

temperature and flow rate and maintained at 40ºC and 0.5ml/min respectively. DAD detector

used over a range of 200-400nm. Mass spectrometry operated in negative ionisation mode.

Silybin metabolites SilybinA7-O-glucuronide and SilybinB 7-O-glucuronide eluted at

15.5min while Silybin A 20-O-glucuronide and SilybinB 20-O-glucuronide obtained at

15.9min and SilybinA 5-O-glucuronide and Silybin B 5-O-glucuronide eluted at 17.4min.

These all metabolites have a same m/z of 657 which show a characteristic fragmentation

pattern of glucuronide metabolite. Minor glucuronide metabolite detected but not isolated this

metabolite detected at 12.3min with m/z of 833 indicates the diglucuronides metabolite of

silybin(Charrier, Azerad et al. 2014).Salvianolic acid is a water-soluble active constituent of

Salvia miltiorrhiza Bunge (Danshen).Salvianolic acid used in the ischemic hemisphere to


1509


improve regional cerebral blood flow by inhibiting cerebral oedema also inhibit the platelet

aggregation. Metabolites of salvianolic acid separated using BDS HYPERSIL C18 column

with dimensions 150mm x 2.1mm, 1.7µm. The mobile phase used is a (solvent A)

acetonitrileand (solvent B) water containing 0.1% formic acid in gradient elution as 80% of B

for 0-2min, a linear decrease of 80-20% of B for 2-4min and 80% of B for 4-7min. Column

temperature and flow rate set is 30ºC and 1ml/min respectively. Salvianolic acid glucuronide

metabolite confirms by reacting with β- glucuronidase enzyme. Salvianolic acid glucuronide

eluted 4.8min at with m/z of 669.19. MS operated in negative ionisation mode. Data

collection and analysis were done by using HPLC-MS Solution version 3.0 software(Han,

Zheng et al. 2012).

4. Application of Glucuronidation

Different UGT enzyme helps to covert the non-polar endogenous substances to a more

hydrophilic derivative which helps to eliminate from the cells or organism. These

glucuronide metabolites are less toxic compared to parent compounds. Glucuronidation helps

to maintain the physiological level of most of the endogenous substances in an

organism(Mazerska, Mroz et al. 2016).An Example is human MRP2 (multidrug resistance

associated protein) efflux transporter which is localised on renal tubular brush border

membrane and hepatic canalicular membrane are excretedin urine (Sallustio 2008).

Glucuronidation is a crucial metabolic pathway in the detoxification of tobacco-specific

nitrosamine i.e.4 -(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (Chen, Dellinger et

al. 2008).Glucuronidation not only helps in detoxification but also helps inactivation of drugs like

morphine to the corresponding Glucuronide used to cure pain.Thiocolchicoside produce active

metabolite 3-O-glucuronidated aglycone used as myorelaxant. Ezetimibe converts to its active

phenolic glucuronide metabolite used as cholesterol absorption inhibitor. Other drugs include

digitoxin, digoxin, α-hydroxy midazolam, retinoic acid and retinol are converted to corresponding

active glucuronide metabolites to give pharmacological beneficial metabolites(Shipkova and

Wieland 2005),(Sallustio 2008). In neonates, chloramphenicol impaired glucuronidation may lead to

major drug toxicity issue (Miners and Mackenzie 1991). Mycophenolic acid acyl-glucuronide binds

to more than one receptor which results in new biological effect (Shipkova and Wieland 2005).In

the aerodigestive tract glucuronidation play a crucial role in the detoxification of

benzo[a]pyrene (BaP) metabolites(Zheng, Fang et al. 2002). Acetaminophen high doses

produces a toxic quinone imine metabolite which causes hepatotoxicity. Administration of

glucuronide inducer 3-methylcholanthrene increases the glucuronide metabolic rate and helps


1510


in detoxification (Kessler, Kessler et al. 2002). Glucuronidation lowers the hepatic bile acid

toxicity and also enhances their urinary excretion and hence protect the liver(Perreault,

Białek et al. 2013).

4. CONCLUSION

Glucuronidation is the main important metabolic process in humans. It helps in detoxification

and elimination of various drugs. After completion of the glucuronidation metabolism based

on the link of glucuronic acid to C, O, N, S atoms it forms C-glucuronides, O-glucuronides,

N-glucuronides and S glucuronide metabolites. UGT enzymes mainly classified as four

families like UGT1, UGT2, UGT3 and UGT8, in these isoforms of UGT enzymes UGT1A2,

UGT2B13 and UGT2B16 present only in rats and rabbits. Polymorphism of UGT enzymes

leads to various diseases. Characterisation and identification of different UGT enzymes along

with metabolites requires since it play vital role in maintaining physiological balance.

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