Novel Heterogeneous Fenton's-Like Catalysis for Degradation of Colchicine Coupled with Extraction of Its Biologically Active Metabolite

Article abstract of DOI:10.1016/j.molliq.2019.111870

Nowadays, drug pollution; a form of water pollution caused by some pharmaceuticals and their metabolites resulting from consumers, industry and hospitals was reported. Colchicine (CLN) is considered one of the pharmaceutical wastewater contaminants which are not eliminated completely in municipal sewage treatment plants and are discharged into receiving water. Due to the higher toxicity of CLN, a novel heterogeneous Fenton's-like catalysis was established for complete degradation of CLN. So, a highly sensitive and specific liquid chromatographic method with quadrupole mass spectrometry (LC/Q-MS) was developed and validated for estimation of CLN in its pure form and in the presence of its degradation product. Herein, GraceSmart RP C18 column was utilized for separation of the cited drug (Retention time t_R = 5.578 min) using methanol: water (55: 45, v/v) at 1.0 mL min^?1. Detection was performed by Agilent 6120 Quadrupole MS detector in a positive ionization mode. Thereafter and for the first time, degradation of CLN by heterogeneous Fenton's-like catalysis using modified polyacrylonitrile (PAN) as a catalyst with H₂O₂ in aqueous acidic medium was performed. This process was firstly optimized by HPLC/UV detection at 248 nm using the aforementioned chromatographic conditions. As a result, CLN degraded completely within 30 min. The only observed degradation product was the biologically active, potent and less toxic antitumor metabolite of CLN (3- demethyl CLN) which was collected, extracted, and analyzed by Fourier Transfer- Infrared Spectroscopy (FTIR) and ¹³Carbon- Nuclear Magnetic Resonance (¹³C NMR). Finally, this method is eco-friendly and complies with the requirements of the green chemistry. It is suitable for complete removal of CLN and/or its metabolite contaminants from wastewater samples and estimation of the target drug without any interference from its degradation products. However, further study is required to expand the method applicability to the pharmaceutical wastewater treatment as well the production of 3- demethyl CLN on a large scale.

Full text of DOI:10.1016/j.molliq.2019.111870

Journal Pre-proof
Heterogeneous Fenton's-like catalysis for degradation of colchicine coupled with
extraction of its biologically active metabolite
Noha M. Hosny, Katherine Huddersman, Noha N. Atia, Samia M. El-Gizawy
PII:
S0167-7322(19)33628-1
DOI:
https://doi.org/10.1016/j.molliq.2019.111870
MOLLIQ 111870
Reference:
To appear in: Journal of Molecular Liquids
Received Date: 29 June 2019
Revised Date: 28 September 2019
Accepted Date: 1 October 2019
Please cite this article as: N.M. Hosny, K. Huddersman, N.N. Atia, S.M. El-Gizawy, Heterogeneous
Fenton's-like catalysis for degradation of colchicine coupled with extraction of its biologically active
metabolite, Journal of Molecular Liquids (2019), doi: https://doi.org/10.1016/j.molliq.2019.111870.
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2019 Published by Elsevier B.V.

Heterogeneous Fenton's-Like Catalysis for
Degradation of Colchicine Coupled with Extraction of
Its Biologically Active Metabolite
1
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a, b*
b
a
Noha M. Hosny
, Katherine Huddersman , Noha N. Atia , Samia M.
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a
El-Gizawy
a
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Assiut
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University, Assiut 71526, Egypt.
b
School of Pharmacy, Faculty of Health and Life Sciences, De Montfort University,
Leicester, UK.
*
Corresponding author: Noha M. Hosny
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E-mail: phar_nmh2011@yahoo.com, noha.hosni@pharm.au.edu.eg,
Tel: +2-01144438525
-
Funding sources: This research did not receive any specific grant from funding
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agencies in the public, commercial, or not-for-profit sectors.
-Declarations of interest: none.
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Abstract
1
2
Nowadays, drug pollution; a form of water pollution caused by some pharmaceuticals
and their metabolites resulting from consumers, industry and hospitals was reported.
Colchicine (CLN) is considered one of the pharmaceutical wastewater contaminants
which are not eliminated completely in municipal sewage treatment plants and are
discharged into receiving water. Due to the higher toxicity of CLN, a novel
heterogeneous Fenton's-like catalysis was established for complete degradation of
CLN. So, a highly sensitive and specific liquid chromatographic method with
quadrupole mass spectrometry (LC/Q-MS) was developed and validated for
estimation of CLN in its pure form and in the presence of its degradation product.
Herein, GraceSmart RP C18 column was utilized for separation of the cited drug
3
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5
6
7
8
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(
Retention time t = 5.578 min) using methanol: water (55: 45, v/v) at 1.0 mL/min.
R
Detection was performed by Agilent 6120 Quadrupole MS detector in a positive
ionization mode.
Thereafter and for the first time, degradation of CLN by heterogeneous Fenton's-like
catalysis using modified polyacrylonitrile (PAN) as a catalyst with H O in aqueous
2
2
acidic medium was performed. This process was firstly optimized by HPLC/UV
detection at 248 nm using the aforementioned chromatographic conditions. As a
result, CLN degraded completely within 30 min. The only observed degradation
product was the biologically active, potent and less toxic antitumor metabolite of
CLN (3- demethyl CLN) which was collected, extracted, and analyzed by Fourier
1
3
Transfer- Infrared Spectroscopy (FTIR) and Carbon- Nuclear Magnetic Resonance
1
3
(
C-NMR).
Finally, this method is eco-friendly and complies with the requirements of the green
chemistry. It is suitable for complete removal of CLN and/or its metabolite
contaminants from wastewater samples and estimation of the target drug without any
2

interference from its degradation products. However, further study is required to
expand the method applicability to the pharmaceutical wastewater treatment as well
the production of 3- demethyl CLN on a large scale.
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Keywords
Colchicine; 3- demethyl colchicine; Liquid chromatography-mass spectrometry;
Fenton's-like catalysis; Drug pollution.
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1
. Introduction
1
Nowadays, drug pollution; a form of water pollution caused by some pharmaceuticals
and their metabolites resulting from consumers, industry and hospitals was reported.
Pharmaceutical pollutants are not eliminated completely in municipal sewage
treatment plants and are discharged into receiving water [1-4]. Drug pollution comes
simply from the excreted drugs and/or their metabolites in the urine in addition to the
expired or unneeded drugs that are flushed unused down the toilet [3]. Other sources
of such pollution include agricultural runoff (because of antibiotic use in livestock)
and pharmaceutical manufacturing [1, 3].
2
3
4
5
6
7
8
9
Filtration to remove course solids followed by biological treatment and sand filtration
are the common processes used for wastewater treatment. Also, Fenton's reagent
10
11
12
13
14
2
+
(
H O with Fe as a catalyst) a form of Advanced Oxidation Process (AOP) is widely
2 2
utilized to oxidize organic water pollutants to carbon dioxide and water by a powerful
hydroxyl radical as following [3, 5];
2
+
3+
−
Fe + H O → Fe + OH (Hydroxyl radical) + OH
15
16
17
2
2
3
+
2+
+
Fe + H O → Fe + OOH (Hydroperoxyl radical) + H
2
2
R (organic substance) + OH → Oxidation products
But the main limitations of classical Fenton's oxidation are pH modulation, the
presence of the catalyst in the final treated samples and the production of sludge
formed by the precipitation of Iron (III) oxo/hydoxo species which are insoluble at the
pH of the process. This sludge is also likely to be contaminated by non-degraded
pollutant [6].
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26
To overcome these demerits, a heterogeneous form of the classical Fenton's catalyst
3
+
incorporating Fe cations in a Fenton's- like catalysis was designed by using
modified polyacrylonitrile (PAN) fibres [7-8]. This modification process involves the
impregnation of catalytically active transition metal complexes on the functionalized
4

surface of PAN fibres. Transition metals are preferred because of their partially filled
d orbitals making them highly reactive [9]. Studies at De Montfort University has led
1
2
3
+
to further development of a novel PAN catalyst through the immobilization of Fe on
the surface of the PAN. The presence of chemically highly reactive nitrile groups on
the polymer allowed functionalization with a mixture of hydroxylamine and hydrazine
salts in alkaline solution to form chelating ligands (carboxylate, amides and oxime)
3
4
5
6
[
8, 10]. This indigenous catalyst has been successfully tested for the oxidative
7
decomposition of several waste effluents from pharmaceutical, textile, agrochemical,
and other model compounds at laboratory scale in a heterogeneous Fenton-like
process using H O as an oxidant in a similar cyclic redox mechanism to the
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2
2
aforementioned where the produced hydroxyl radical reacts with the organic pollutant
to form oxidation products [11-12].
Colchicine (CLN) is considered one of the pharmaceutical wastewater contaminants
[
1, 2, 13]. CLN is an alkaloid obtained from different Colchicum species (Figure 1). It
is an official drug in the European, Chinese, Japanese, and International
Pharmacopeias as well USP. It is commonly used to relieve pain and inflammation
associated with acute gouty attacks by decreasing the leucocyte mobility [14]. CLN
also has an antimitotic action and is widely used in treatment of amyloidosis, Behçet’s
syndrome, familial Mediterranean fever, idiopathic thrombocytopenic purpura,
pericarditis, primary biliary cirrhosis, and pyoderma gangrenosum [15]. Nausea,
diarrhea, vomiting and abdominal pain associated with CLN antimitotic action are its
main side effects and the first signs of its toxicity. With CLN overdose; anemia,
multiple organ failure, bone marrow depression, hepatocellular with muscle damage,
renal damage and CNS toxicity have been reported [13-15].
5

Furthermore, CLN derivatives such as 3-demethyl CLN (Figure 1), 2-demethyl CLN,
colchicoside and thiocolchicoside have been reported for their possible application in
management of certain forms of leukemia and solid tumors [16]. Unlike CLN, they
have potent anti-inflammatory and anti-tumor activity with lower toxicity. But, due to
rarity of CLN derivatives through CLN-producing plants, many trials have been made
to find alternative routes for their industrial-scale production [16-19].
Biotransformation of CLN employing CYP3A4 enzyme or microbial demethylation
were reported for CLN demethylation [17-19].
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3
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8
Concerning literature, liquid chromatography (with UV, MS or Tandem MS
detection) [20-28], gas chromatography [29-30], thin layer chromatography (TLC)
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[
31-33], fluorimetric and spectrophotometric methods [34-37] have been reported for
estimation of CLN alone or combined with other drugs.
The present work is the first study that focuses on the application of heterogeneous
Fenton's-like catalysis for fully degradation of CLN using the modified PAN catalyst
with H O in aqueous acidic medium. So, a highly sensitive and specific liquid
2
2
chromatographic method with quadrupole mass spectrometry (LC/Q-MS) for
estimation of CLN in pure form and in the presence of its degradation product was
developed and validated.
Thereafter, the biologically active, potent and less toxic antitumor derivative of CLN
(3- demethyl CLN) produced during CLN degradation was collected, extracted, and
1
3
analyzed by Fourier Transfer- Infrared Spectroscopy (FTIR) and Carbon- Nuclear
1
3
Magnetic Resonance ( C-NMR).
This work was designed to fulfill the principles required for the green chemistry [38].
So, hazards from chemical toxicity and waste reduction in addition to discovering
replacements for hazardous substances were our main targets.
6

2
2
. Experimental
.1. Apparatus
1
2
Agilent 6120 Quadrupole LC/MS system linked to 1260 Infinity auto-sampler,
solvent manager, pump, thermostated column chamber and Quadrupole MS detector
3
4
(
Agilent Technologies, USA) was used. This system was operated with OpenLAB
CDS ChemStation Edition C. 01. 06 software. PerkinElmer Series 200 HPLC System
Model LCTURBO, Serial no. ZAAA0646, PerkinElmer® Inc., USA) connected to
5
6
(
7
series 200 degasser, pump and UV/VIS detector was utilized for optimization of the
drug degradation process. A rheodyne injection valve (Model 7725i, USA) with a 20
8
9
ꢀ
L loop and 100 ꢀL Hamilton – Bonaduz, Schweiz (Switzerland) sample syringe
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23
were used for sample application. The HPLC system was operated with TotalChrom
Workstation version 6.3.4 software. The GraceSmart RP C18 encapped column (250
mm length x 4.6 mm inner diameter, 5 µm particle size, 120 Å pore size) was coupled
to either LC/MS or HPLC/UV system.
ALPHA FTIR (Serial no. GI004911, operated with OPUS Spectroscopy Software)
TM
and AVANCE - UltraShield 400 NMR (Serial no. GH007300) spectrometers
(BRUKER UK Limited, UK) in addition to Thermo Scientific-Evolution 220 UV-VIS
spectrophotometer with 1 cm quartz cuvettes (Waltham, Massachusetts, USA) were
also used. Moreover, analytical series Fisher-Scientific balance-PAS214C (China),
Jenway 350 pH meter (China) and ultrasonic cleaner- Kerry PUL 125 (England) were
utilized. Furthermore, Nylon 66 membranes (0.45 µm pore size, 47.0 mm diameter)
and Fisherbrand PTFE syringe filters (0.45 µm) were obtained from SUPELCO, USA
and Fisher Scientific Co., UK; respectively.
7

Regarding the catalytic oxidation process, Carousel 6 Plus Reaction Station™ set
Radleys, UK) consists of stirring hotplate and 250 mL-round bottom flasks with
1
(
2
reflux head was used.
3
Interflon glass gravity column (200 mm length x 20 mm inner diameter, Sigma-
Aldrich Co., UK) and UVGL-55 Handheld lamp (USA) were utilized during
4
5
®
extraction of the degradation product. Also, Quantofix peroxide semi-quantitative
6
test strips (Macherey-Nagel Germany) and Büchi Rotavapor R-300 (UK) were used.
7
2
.2. Chemicals and Solvents
CLN (≥95%) was purchased from Sigma-Aldrich Co., UK. LC/MS grade methanol
99.95%) and water (99.9%) were purchased STRATLAB Laboratory Supplies, UK.
8
9
(
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HPLC grade Hydrogen peroxide (30% w/w), analytical grade hydrochloric acid
TM
(
36%), sodium hydroxide, FeSO .7H O and Acros Organic deuterated chloroform
4 2
for NMR were bought from Fisher Scientific Co., UK.
Modified PAN catalytic mesh was supplied by School of Pharmacy, De Montfort
University, Leicester, UK.
TLC aluminium sheets precoated with silica gel G 60F254 plates (Fluka, 5 x 5 cm, 0.20
mm layer thickness) and Silica gel for column chromatography (200-400 mesh, 60 Å)
were obtained from Sigma-Aldrich Co., UK.
2
.3. Standard Solutions preparation.
5
0.0 mg of CLN were dissolved in 50.0 mL water to obtain the stock standard
-1
solution (1.0 mg mL ). This solution was kept in a refrigerator away from light.
-1
The stock solution was firstly diluted with water to prepare 200 ng mL followed by
serial dilution with the same solvent to prepare the working standard solutions (0.1-50
-1
ng mL ).
2
5
8

2
2
1
.4. Analytical procedures.
1
.4.1. General Chromatographic Conditions.
2
0 µL of the working standard or sample solution of the studied drug was injected
3
automatically by the autosampler on Agilent 6120 Quadrupole LC/MS system linked
to GraceSmart RP C18 column (250 mm length x 4.6 mm inner diameter, 5 µm
particle size, 120 Å pore size; Controlled temperature at 25± 0.8°C). Then, isocratic
4
5
6
-1
elution was carried out by methanol: water (55:45, v/v) at flow rate 1.0 mL min . The
7
total run time was 10 minutes.
8
Detection was performed by Agilent 6120 Quadrupole MS detector in a positive
9
ionization mode (Drying gas: N at 12.0 L/min; its Temp. 350°C, Capillary voltage
10
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18
19
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22
2
3
000 V, and the main settings: Rough Vacuum 1.38 Torr, Quadrupole Temp. 100°C,
Nebulizing Pressure 35 psig, TurboSpd 100%, Fragmentor 70, Gain 1.0, Threshold
50). MS data were collected as either total ion current (TIC, m/z 50-500), or selected
1
ion monitoring (SIM) at m/z 400.4. CLN mass spectrum showed a prominent
+
molecular ion [M+H] peak at m/z= 400.2.
2
.4.2 Construction of Calibration Curves.
Working standard solutions of CLN equivalent to (0.10, 1.0, 10.0, 20.0, 30.0, 40.0 and
-1
5
0.0 ng mL ) were prepared in water. Then, the assay was done as mentioned under
the general chromatographic conditions (Section 2.4.1).
The peak area values (µV*Sec) were plotted against the drug final concentration (ng
-1
mL ) to get the calibration graph. Thereafter, the corresponding regression equation
was calculated.
2
2
2
3
4
5
9

2
2
.5. Degradation of Colchicine.
1
.5.1. Preparation of PAN Catalyst.
2
To remove excess iron from the PAN catalyst, washing of the mesh was performed by
scrubbing and immersing it in tap water. The colour of water changed from clear to
brown which shows leaching off the excess iron from the surface of the catalyst. This
process was done until there was no further colouration. The washed PAN was cut to
pieces (20*20 cm) and transferred into a beaker with double distilled water then pH
was adjusted at 3.0 using 0.1 N hydrochloric acid and/or sodium hydroxide. Upon
stabilization of pH (3.0), the PAN mesh was removed away from water and air-dried
at room temperature.
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2
.5.2. Procedure for CLN Degradation.
-1
Working standard solution of CLN (50.0 µg mL ) was prepared in acidic double
distilled water (pH 3.0; adjusted by 0.1 N HCl and/or NaOH). 100 mL of this working
solution were transferred into a carousel flask followed by addition of 8 grams of
2
PAN catalyst (cut into 2 cm pieces) and a magnetic stirrer flea. pH of the flask
content was checked and readjusted to the desired value (3.0). Carousel 6 Plus
Reaction Station™ stirring hotplate was set to 500 rpm at 25°C to ensure a proper
mixing (Figure S1). Thereafter, the catalytic reaction was initiated by addition of 100
µL of 30% H O (corresponding to 333 ppm) to the flask content (CLN+ PAN).
2
2
Samples were taken at predetermined time intervals of 5 min to monitor the
degradation of CLN (the targeted drug degraded completely within 30 min). The first
sample (0 min; CLN only) was taken before the introduction of the catalyst.
Subsequently, the catalyst and H O were added and the samples were pulled every 5
2
2
min and filtered through PTFE syringe filters. 10 µL of the collected samples were
then injected on LC/MS system. The assay was then carried out in triplicates as
1
0

mentioned before (Section 2.4.1). MS data were collected as either TIC at m/z 50-500
or SIM at m/z 400.4 and 386.4 for CLN and its degradation product; respectively.
1
2
+
Mass spectra showed two prominent molecular ion peaks [M+H] at m/z= 400.2 and
3
3
86.1 for CLN and demethylated-CLN; respectively..
4
Blank experiment was done in the same manner excluding the drug.
Furthermore, two experiments were performed similarly to evaluate the CLN stability
in the absence of the PAN catalyst (The first was for CLN in acidic water; while the
second one was for CLN in presence of H O at pH 3.0).
5
6
7
8
2
2
2
.5.3. Isolation of the degradation product.
9
During the degradation process, CLN degraded to its biologically active, potent and
less toxic antitumor metabolite (3- demethyl CLN). It reached a maximum
concentration within 5 min of the catalytic oxidation reaction. Due to the product
medical importance, we tried to separate and collect it by the aid of TLC and
fractional column chromatographic techniques.
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20
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25
The catalytic oxidation reaction was stopped after 5 min via the removal of PAN
pieces (by filtration) and evaporation of excess H O (by heating on a water bath for 5
2
2
®
min and H O content was checked by Quantofix peroxide semi-quantitative test
2
2
strips). The degraded solution mixture (CLN and its metabolite) was preliminary
separated on silica gel TLC plates using methanol: water (55:45, v/v) and the spots
were visualized by portable UV- lamp at 254 nm (Retardation factor (R ) = 0.61 and
f
0
.13 for CLN and its derivative; respectively).
Subsequently, the fractional column chromatographic method was established to
collect each compound separately. Interflon glass column (200 mm length x 20 mm
inner diameter) was carefully packed with silica gel and conditioned by methanol then
loaded with the degraded solution mixture. Methanol: water (55:45, v/v) was firstly
1
1

used as a mobile phase to elute CLN. Then, 3- demethyl CLN was eluted by 100%
water. The eluted fractions were checked for their purity by TLC plates and UV/VIS
spectrophotometer (Figure S2).
1
2
3
The collected fractions of 3- demethyl CLN were evaporated using Büchi Rotavapor
R-300 to obtain a yellowish- white powder of the desired product.
For structure confirmation, CLN and 3- demethyl CLN samples were analyzed by
FTIR and NMR spectrometers.
4
5
6
7
3
. Results and Discussion
8
The main objective of the presented work is to establish an eco-friendly method for
fully degradation of CLN by heterogeneous Fenton's-like catalysis for the first time
9
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15
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17
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19
20
21
22
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25
(
using modified PAN catalyst with H O in aqueous acidic medium). So, a simple,
2 2
highly sensitive and specific LC/Q-MS method was developed and validated for
determination of CLN in its pure form and in the presence of its degradation product.
Finally, the isolation and identification of the biologically active metabolite of CLN
(3- demethyl CLN) produced during the degradation process were achieved.
3
3
.1. The developed LC/Q-MS Method.
.1.1. Chromatographic Conditions Optimization.
To achieve a good resolution and fast analysis with symmetric peaks, mobile phase
composition and flow rate were tested.
3
.1.1.1. Mobile phase
After testing acetonitrile, methanol and ethanol as organic solvents, a good resolution
and a sharp peak with higher area was observed when methanol was the organic
modifier. While, ethanol and acetonitrile gave asymmetric peak with CLN (broad and
tailed). Also, the percentage of the chosen organic solvent was varied (from 10 % to
9
0 %). Upon increasing the methanol percentage ≥70%, improper resolution between
1
2

CLN and its degredant was observed. On the other hand, good separation was
achieved within longer run time (>10 min) when the methanol content was decreased
1
2
≤
40%. So, the chosen mobile phase (55% methanol to 45% water) gave the best
3
resolution and sharp peak of the target drug in presence of its degradation product
4
within reasonable time (6 min). The retention times (t ) were 5.578 and 4.281 for
5
R
CLN and 3-demethyl CLN; respectively. The LC/MS chromatograms for the cited
6
drug and its degradation product were shown in Figure 2.
7
3
.1.1.2. Flow rate
8
The influence of flow rate on the separation of CLN alone or in presence of its
9
-
1
degradation product was studied in the range of 0.3-2 mL min . In the preliminary
studies, overlapping and poor resolution was observed at higher flow rates ≥1.2. But
at lower flow rates ≤0.8, good resolution was achieved with broad asymmetric peaks
at longer run time (more than 10 min) for the entire elution of these compounds. So,
10
11
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20
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24
-
1
an isocratic mode of flow rate (1 mL min ) was set to enhance the method's
resolution within short separation time (less than 6 min). Thus, the proposed method
can successfully applied for rapid analysis of CLN in the presence of its degredant.
3
.1.2. Method Validation.
As mentioned in ICH guidelines on the validation of analytical methods ICH Q2 (R1),
linearity, detection (LOD) and quantification (LOQ) limits, precision, accuracy,
robustness, selectivity and specificity of the proposed LC/MS were evaluated [39].
3
.1.2.1. Linearity and sensitivity parameters.
According to the optimal chromatographic conditions, six calibration curves for
standard solutions of CLN at seven concentration levels (Figure 3) were constructed
-1
by plotting peak area against drug concentration (ng mL ). The results presented in
1
3

-1
Table 1 indicate good linearity over a range of 0.10 – 50.0 ng mL with an excellent
1
correlation coefficient (0.9999).
2
Also, 3 σ/S and 10 σ/S were used for calculation of LOD and LOQ of the studied
drugs; respectively (σ is the standard deviation of y-intercept of the regression
equation and S is the slope of the calibration curve). The lower LOD and LOQ values
3
4
5
-
1
obtained (16.4 and 49.8 pg mL ; respectively) indicate a high sensitivity of the
proposed method. In conclusion, the method's simplicity and sensitivity are superior
to other reported LC/MS or Tandem MS methods [23-28].
6
7
8
3
.1.2.2. Accuracy and precision
9
-
1
Low, medium, and high concentration ranges (0.100, 20.0, and 50.0 ng mL ) of CLN
were analyzed by the developed LC/MS method. After injection of each
concentration in six replicates, recovery percentages were calculated. The recoveries
were in the range from 100.12 to 100.18% with RSD ≤0.89% indicating good
accuracy of the proposed method (Table 2). Moreover, the three above-mentioned
concentrations were analyzed in triplicates for intra-day precision and gave recoveries
of (99.90-100.34%) and (99.67-100.36%) for inter-day precision (n= 9). RSD values
were found to be ≤ 0.68 for intra- and inter-day precision (Table 2). These results
confirm that the acceptable repeatability and accuracy of the proposed method for
assay of CLN.
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25
3
.1.2.3. Robustness.
Minor changes of the optimal chromatographic parameters were studied. It was found
that the slight alteration in flow rate and mobile system composition had no
significant influence on the method's performance. The results shown in Table 3
indicate the reliability of our method during normal usage.
3
.1.2.4. Specificity and selectivity.
1
4

The proposed method is able to separate the studied drug completely from its
degradation product. After injection of the obtained degraded solution (CLN with its
derivative), clean chromatograms were observed without any interfering peaks from
the catalyst, H O or the degradation product especially at the target retention time
1
2
3
4
2
2
(
Figure 2 & 4). Additionally, the method's specificity was enhanced using MS
detection with SIM at the drug's m/z 400.4.
.2. Optimization of CLN Degradation Reaction.
5
6
3
7
To ensure a complete and efficient degradation process, reaction parameters (PAN
8
catalyst amount, H O volume and the effect of pH) were evaluated using HPLC/UV
9
2
2
system.
10
11
12
13
The procedure of CLN degradation (section 2.5.2) was carried out and the samples
were pulled after 15 min of the reaction time. 20 µL of the sample was then injected
manually on HPLC/UV system. As mentioned before (section 3.1.1), methanol: water
-
1
(
55: 45, v/v) at flow rate (1.0 mL min ) were used as the optimal chromatographic
14
15
16
conditions during this study. The UV detector was set at 248 nm (One of two
maximum wavelengths of CLN; Figure S3).
Figure 4 represents the HPLC - Chromatograms of CLN with and without its
degradation product.
17
18
3
.2.1. PAN Catalyst Amount
19
20
21
22
23
24
25
The procedure for CLN degradation (section 2.5.2) was performed using different
weights of PAN mesh (2, 4, 6, 8, 10 g). It was found that 8 g of PAN catalyst gave the
lowest peak area of CLN and an efficient degradation (Figure 5A).
3
.2.2. Volume of H O2
2
To study the effect of H O concentration on the degradation reaction, different
2
2
volumes (20, 40, 60, 80, 100, 120, 140 µL) of 30% H O were evaluated. As shown in
2
2
1
5

(Figure 5B), the highest amount of CLN (about 92 %) was degraded within 15 min
1
upon using 100 µL of 30% H O (=333 ppm).
2
2
2
On the other hand, calibration curve of H O was constructed by plotting peak areas
3
2
2
against different concentrations of H O (50- 400 ppm) in acidic water (pH 3.0). The
4
2
2
6
obtained linear regression equation was Y= 6088.4 X + 10 ; R² = 0.9945 (Figure S4).
5
We found that ~ 85% of the chosen initial concentration of 30% H O (100 µL) was
6
2
2
consumed on CLN complete degradation. Also, H O scavenges hydroxyl radical
7
2
2
upon the increase of H O concentration.
8
2
2
3
.2.3. Effect of pH
9
Variation of pH of both CLN working standard solution and PAN mesh (during its
preparation) was investigated. Chu et al. [40] found that the oxidation potential of
hydroxyl radicals decreased with increasing pH. Also, degradation rate and efficiency
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2+
decreased at pH below 3.0 due to the formation of iron complex species [Fe(H O) ]
2
6
which reacts more slowly with H O [41]. While; at slightly higher pH >3.0, the
2
2
activity of Fenton's reagent was reduced due to the presence of an inactive iron
oxohydroxides [42]. So, different low pH values (3.0, 3.5, 4.0, 4.5; adjusted by 0.1 N
HCl and/or NaOH) were tested. A higher loss of CLN content was achieved at pH 3.0
(
Figure 5C). Our finding agrees with many previous studies that recommended the
use of pH 3.0 for Fenton’s oxidation [4].
.2.4. Monitoring of Degradation Reaction.
3
To monitor the degradation of CLN, the samples were analyzed by LC/Q-MS under
the optimal reaction parameters at different reaction time (time intervals of 5 min over
0
- 30 min). MS data were collected as either total ion current (TIC, m/z 50-500), or
selected ion monitoring (SIM) at m/z 400.4 and 386.4 for CLN and its demethylated
product; respectively). As a result, CLN was completely degraded within 30 min
1
6

(Table S1). One product was observed during this reaction (3- demethyl CLN; tR=
1
4
.281 min; m/z 386.1). Its highest concentration was reached within 5 min of the
2
reaction time.
3
At 30 min of the reaction time, neither CLN nor its product's peaks were detected
4
(Figure 6). As a result, this method could be applied successfully for the complete
5
removal of CLN and/or its derivative from the wastewater during the treatment
6
process.
7
3
.3. Isolation and Identification of the Degradation Product.
8
Due to the medical importance of the resulting compound (3- demethyl CLN) during
the CLN catalytic oxidation process, we tried to extract and identify it. 3- Demethyl
CLN is more polar than CLN. So, it was separated easily with a simple fractional
column chromatographic technique. Thereafter, its collected dried fractions were
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
TM
characterized by ALPHA FTIR and AVANCE - UltraShield 400 NMR
spectrometers in addition to Thermo Scientific-Evolution 220 UV-VIS
spectrophotometer. The UV spectra (Figure 7A& A') showed a slight blue shift of 3-
demethyl CLN (343 nm) compared to CLN (350 nm) because of the demethylation of
CLN. Furthermore, MS spectrum (Figure 2) showed a signal at m/z 386.1
corresponding to 3- demethyl CLN. For further structure elucidation, a signal of
-1
phenolic hydroxyl group (3657 cm ) was detected on FTIR spectrum of 3- demethyl
1
3
CLN (Figure 7B'). Also, C-NMR spectrum of 3- demethyl CLN (Figure 7C')
confirmed that the absence of methoxy group signal at position 3 (56.42 ppm) as
1
3
compared with the standard CLN spectrum (Figure 7C). Our C-NMR data of CLN
and 3- demethyl CLN that agree with Dubey et al. and Zhang et al. findings [16, 19]
are summarized in Table S2. Our results ensured that hydroxyl radical (produced from
Fenton's- like catalysis) attacks CLN at position no. 3 and not the other positions.
1
7

Methoxy group is an electron-donating making the attached carbons more attractive to
the electron-seeking hydroxyl radical. Attack at C-3 is preferred over attack at C-1 or
C-2 as these sites are sterically hindered [43].
1
2
3
3
.4. Stability of CLN.
4
To evaluate the CLN stability in the absence of PAN catalyst; influence of 100 µL of
H O and/or acidic aqueous medium (pH 3.0; adjusted by 0.1 N HCl) on CLN (50.0
5
6
2
2
µg/mL) were studied using HPLC/UV detection. It was observed that the cited drug
7
was stable in H O and/or acidic medium for at least 30 min without any detected
8
2
2
degradation (Figure 8). On the other hand, CLN disappeared within 30 min in the
presence of PAN catalyst with H O at pH 3.0 (Figure 6).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2
2
4
. Conclusion.
Eco-friendly, highly sensitive and specific liquid chromatographic method with
quadrupole mass spectrometry (LC/Q-MS) was developed and validated for
quantitation of colchicine (CLN) in its pure form and in the presence of its
degradation product. MS data were collected as either total ion current (TIC, m/z 50-
5
00), or selected ion monitoring (SIM) at m/z 400.4 for CLN. A good linearity
(r=0.9999) was achieved over a concentration ranging from 0.1 to 50 ng/mL with
LOD (16.4 pg//mL).
Additionally and for the first time, heterogeneous Fenton's-like catalysis was
established for degradation of CLN using modified polyacrylonitrile (PAN) catalyst
with H O at pH 3.0. As a result, CLN degraded completely within 30 min.
2
2
Thereafter, the biologically active, potent and less toxic antitumor metabolite of CLN
3- demethyl CLN) produced during CLN degradation was collected, extracted, and
(
1
3
analyzed by Fourier Transfer- Infrared Spectroscopy (FTIR) and Carbon- Nuclear
1
3
Magnetic Resonance ( C-NMR).
1
8

Finally, this work is the first study developed for complete removal of CLN from the
water samples and production of less toxic and expensive antitumor drug (3- demethyl
CLN). This work has important environmental and pharmaceutical impact to
wastewater treatment from pharmaceutical pollutants as well the production of an
expensive antitumor drug at the same time. It also complies with the requirements of
green chemistry.
1
2
3
4
5
6
However, further study is required to expand the method applicability on the
pharmaceutical wastewater treatment as well the large scale production of 3-
demethyl CLN.
7
8
9
Conflict of Interest:
10
11
12
13
14
15
16
"There are no conflicts to declare"
Acknowledgment:
The corresponding author thanks the Egyptian Ministry of Higher Education and
Scientific Research in addition to the Egyptian Cultural Affairs & Mission Sector for
granting her a short-term mission to De Montfort University, Leicester, the United
kingdom to carry out this work.
1
1
1
2
2
2
2
2
2
7
8
9
0
1
2
3
4
5
1
9

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2
2
2
2
2
1
2
3
4
5
2
5

Figure captions
1
2
Figure 1. Chemical structures of the investigated drug and its derivative.
Figure 2. Total ion current (TIC) chromatograms and mass spectra of CLN (A & A')
and its degradation product (B & B').
3
4
Figure 3. Three dimensional diagram of LC/MS Chromatograms of CLN in seven
5
6
-1
concentration levels (0.10 – 50.0 ng mL ); three replicates for each concentration.
-1
Figure 4. HPLC - Chromatograms of (A) standard solution of CLN (50.0 µg mL ),
B) CLN with its degradation product (after 5 min of the reaction time), (C) 1.0
mg/mL FeSO .7H O solution, (D) PAN catalyst and (E) PAN catalyst with H O all
7
(
8
9
4
2
2
2;
solutions were prepared at pH 3.
10
Figure 5. Effects of (A) PAN catalyst amount, (B) the volume of 30% H O and (C)
11
12
13
2
2
-1
pH on the degradation of CLN (50.0 µg mL ) (Samples were measured after 15 min
of the reaction time).
-1
Figure 6. Influence of the reaction time (0-30 min) on CLN (50.0 µg mL )
14
15
degradation.
Figure 7. Structure elucidation of CLN (A
,
B & C) and 3- demethyl CLN (A'
,
B' &
16
17
1
3
C') using UV
, FTIR and C-NMR; respectively.
-
1
Figure 8. The stability of 50.0 µg mL CLN in the presence of 100 µL of 30% H O
18
19
2
2
and/or acidic aqueous medium (pH 3.0); with/without PAN catalyst.
2
2
2
2
0
1
2
3
2
2
4
5
2
6

Table 1. Optimum chromatographic conditions and quantitative parameters of the
proposed LC/MS method for determination of CLN.
1
2
Studied drug
Mobile phase
CLN
Methanol: Water (55: 45, v/v)
Retention time; t (min)
5.578
0.1 – 50
0.9999
0.9997
R
-1
Linearity range (ng mL )
Correlation coefficient (r)
2
Determination coefficient (R )
a
5
Intercept (a) ± SD
1.50×10 ± 728.97
a
5
Slope (b) ± SD
1.46×10 ± 89.27
b
LOD
16.4
49.8
c
LOQ
a
Average of six replicates.
3
4
5
6
7
b
-1
Limit of detection (pg mL ).
c
-1
Limit of quantitation (pg mL ).
8
9
1
1
1
1
1
0
1
2
3
4
2
7

Table 2. Accuracy, Intra-day and Inter-day precision of the developed LC/MS method for
1
2
determination of CLN.
Authentic Concentration
Accuracy
Intra-day precision
Inter-day precision
-1
drug
(ng mL )
%
Recovery RSD % % Recovery
RSD % % Recovery
RSD %
±
SD*
± SD**
± SD***
0
.10
20.0
0.0
100.15 ± 0.84
100.18 ± 0.67
100.12 ± 0.89
0.84
0.67
0.89
100.34 ± 0.62
0.62
0.34
0.12
100.36 ± 0.68
100.29 ± 0.40
99.67 ± 0.22
0.68
0.40
0.22
CLN
9
9.90 ± 0.34
5
1
00.24 ± 0.12
*
*
Average of six replicates.
*Average of three replicates.
**Average of nine replicates.
3
4
*
-
5
6
Results are calculated from standard curve.
7
8
9
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
2
8