International Journal of PharmTech Research

CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563

Vol.12, No.03, pp 08-21, 2019

Validated Analytical Method for the Determination of

Sorafenib in Dosage form and Human plasma in presence of
it Degradation Products
Wael Talaat1*, Mohamed M.Y. Kaddah2
1
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy,
Damanhour University, Damanhour 25111, Egypt
2
Pharmaceutical and fermentation development center, City of scientific research
and technology applications, New Borg El-Arab 21943, Alexandria, Egypt

Abstract : Herein, the potency, bioavailability and purity of sorafenib can be easily investigated
in the presence of different degradation products through the present work. The bioanalysis of
sorafenib in tablets and human plasma was achieved by a simple chromatographic procedure.
The separation was conducted at room temperature using a stainless steel Hibar C 18 (150 X 4.6
mm i.d ). The analytes were detected with UV detector at 255 nm. A simple mobile phase of
acetonitrile / phthalate Buffer / methanol (75: 24.5: 0.5, v/v) (pH 4) was eluted at a flow rate of
1.5 mL/ min. A rectilinear calibration curve was obtained over concentration range of 0.05 –
2.0 µg /mL, with a detection and quantification limits (LOD, LOQ) of 0.006 and 0.017 µg /mL
respectively.
Key words : Bioanalysis, HPLC, Stability indicating , Kinetics, tablets, real plasma.

1. Introduction
Sorafenib (Fig 1) is 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl] carbamoyl amino] phenoxy]-N-methyl-
pyridine-2-carboxamide 1. Sorafenib is tyrosine protein kinases inhibitor. It is used for the treatment of
hepatocellular, renal cell and thyroid carcinoma 2. There is no official method for sorafenib determination. It has
been analysed by different HPLC methods using different mobile phases like acetonitrile / water (82.5 : 17.5,
v/v)3, 20 mM potassium dihydrogen phosphate / acetonitrile (35:65, v/v) 4 . 20mM ammonium acetate /
acetonitrile / methanol (2.5:6.7:8.3%) 1 , acetonitirile / 10mM ammonium formate (54:46, v/v)5, gradient elution
using formic acid in water / acetonitrile 6, acetonitrile / 20mM ammonium acetate (40:60 v/v)7, acetonitrile /
0.1% formic acid in water (65/35 (v/v)) 8 , acetonitrile/10 mM ammonium acetate (65:35, v/v) containing 0.1%
formic acid 9. Our proposed method is more sensitive than the others as it can measure down to 0.05 μg/mL

Wael Talaat et al /International Journal of PharmTech Research, 2019,12(3): 08-21.

DOI: http://dx.doi.org/10.20902/IJPTR.2019.120302
 Journal of Chromatography A 1645 (2021) 462095

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Bio-activation of simeprevir in liver microsomes and characterization

of its glutathione conjugates by liquid chromatography coupled to
ultrahigh-resolution quadrupole time-of-flight mass spectrometry
Mohamed M.Y. Kaddah a,∗, Susan Billig b, Ramona Oehme b, Claudia Birkemeyer b
a
Pharmaceutical and Fermentation Industries Development Center, City of Scientific Research and Technological Applications, New Borg El-Arab 21934,
Alexandria, Egypt
b
Research Group of Mass Spectrometry, Faculty of Chemistry and Mineralogy, University of Leipzig, Linnèstr. 3, 04103 Leipzig, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Liquid chromatography coupled to a triple quadrupole and, alternatively, to an ultrahigh-resolution
Received 30 September 2020 quadrupole time-of-flight (UHR-QqTOF) mass spectrometers was used to collect qualitative and quanti-
Revised 23 February 2021
tative information from incubations of the anti-hepatitis C drug simeprevir with human and rat liver mi-
Accepted 19 March 2021
crosomes, respectively, supplemented with NADPH and glutathione. For this, different chromatographic
Available online 25 March 2021
methods using two different chromatographic columns, Kinetex® 2.6 μm C18 (50 × 3 mm) and Atlantis
Keywords: T3 (100 Å, 3 μm, 4.6 mm × 150 mm), have been employed. For determination and structural character-
Ultrahigh-resolution quadrupole ization of the reactive metabolites, we used information obtained from high-resolution mass spectrom-
time-of-flight (UHR-QqTOF) etry, namely accurate mass data to calculate the elemental composition, accurate MS/MS fragmentation
Broadband collision induced dissociation patterns for confirmation of structural proposals, and the high mass spectral resolution to eliminate false-
(bbCID) positive peaks. In this study, the use of high-resolution mass spectrometry (HR-MS) enabled the identi-
Triple quadrupole mass spectrometer
fication of 19 simeprevir metabolites generated by O- respectively N-demethylation, oxidation, dehydro-
Simeprevir
Reactive metabolites
genation, hydrolysis, and formation of glutathione conjugates. The in silico study provides insights into the
Hepatotoxicity sites of simeprevir most amenable to reactions involving cytochrome P450 . The developed methods have
been successfully applied to analyze simeprevir and its metabolites simultaneously; based on this data,
potential metabolic pathways of simeprevir are discussed. In general, the obtained results demonstrate
that simeprevir is susceptible to form reactive simeprevir-glutathione adducts and cyclopropansulfon-
amide, which may explain the implication of simeprevir in idiosyncratic adverse drug reactions (IADRs)
or hepatotoxicity.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction in human plasma [4], or simultaneously with other direct-acting

antivirals such as elbasvir, grazoprevir, sofosbuvir, ledipasvir, da-
Simeprevir is a direct-acting NS3/4A serine protease inhibitor clatasvir, and velpatasvir [5–7], but not for simeprevir with its
utilized with other antiviral drugs in the remediation of chronic metabolites.
infections with hepatitis C virus (HCV) of genotypes 1 and 4 [1]. It The term ‘’drug metabolism’’ includes metabolic pathways that
has been associated with isolated, uncommon cases of mild, acute alter the chemical structure of drugs. These pathways are a form
liver laceration during treatment and to incidental cases of hep- of enzymatic biotransformation and can be divided into two major
atic decompensation in patients with a recorded history of cirrho- categories: phase I (biotransformation) and phase II (conjugation)
sis [2]. However, the reasons for these incidences are yet unknown. pathways [8]. In phase I, a drug is either activated or inactivated by
Such reactions are often related to the modification of a drug dur- a variety of enzymes involving the formation of a functional group
ing metabolization in the target organism [3], but only little data or the modification of an existing one by oxidation, reduction, or
are available for simeprevir within this context. Only a few liquid hydrolysis. In the subsequent phase II reactions, the drug and/or
chromatography tandem-mass (LC-MS/MS) spectrometry methods its metabolites are coupled to an endogenous conjugating molecu-
are reported in the literature for the determination of simeprevir lar species such as glucuronic acid, sulfate, glycine, etc. [9,10]. The
presence of carboxyl or hydroxyl groups in the parent compound
∗
increases its affinity to form sodium adducts under electrospray
Corresponding author.
E-mail address: mkaddah@srtacity.sci.eg (M.M.Y. Kaddah).
conditions, and consequently, its metabolites (phase I & phase II)

https://doi.org/10.1016/j.chroma.2021.462095
0021-9673/© 2021 Elsevier B.V. All rights reserved.
Received: 15 January 2021 Revised: 11 April 2021 Accepted: 19 April 2021
DOI: 10.1002/bmc.5146

RESEARCH ARTICLE

Determination and structural characterization of ravidasvir

metabolites by LC coupled to triple quadrupole linear ion trap
MS: Application to pharmacokinetics and phase I metabolism
in rats

Mohamed Mohamed Yousri Kaddah1 | Wael Talaat2 | Maha A. El Demellawy3

1
Pharmaceutical and Fermentation Industries
Development Center, City of Scientific Abstract
Research and Technological Applications, New Hepatitis C virus (HCV) is an infectious disease that has become a global clinical
Borg El-Arab, Alexandria, Egypt
2 issue because of its significant morbidity and mortality. Novel anti-hepatitis C drugs
Department of Pharmaceutical Analytical
Chemistry, Faculty of Pharmacy, Damanhour are continuously developed to decrease the pervasiveness of the infection globally.
University, Damanhour, Egypt
A synthetic ravidasvir, benzimidazole-naphthylene-imidazole derivatives, has been
3
Center of Excellence for Preclinical Research
in Drug Development, City of Scientific used as an anti-HCV drug. This study determined the metabolites of ravidasvir and
Research and Technological Applications, New its pharmacokinetics in rats using information-dependent acquisition and multiple
Borg El-Arab, Alexandria, Egypt
reaction monitoring scanning modes in linear ion trap LC–MS/MS instrument,
Correspondence respectively. Two time-programming linear-gradient chromatographic methods were
Mohamed Mohamed Yousri Kaddah,
Pharmaceutical and Fermentation Industries employed using a Kinetex C18 column (50
3 mm, 2.6 μm) and a Luna HILIC
Development Center, City of Scientific column (100
4.6 mm, 3 μm) for the qualitative and quantitative determination of
Research and Technological Applications, New
Borg El-Arab 21934, Alexandria, Egypt. ravidasvir and its metabolites, respectively. In silico prediction where sites in a
Email: mkaddah@srtacity.sci.eg molecule are susceptible to metabolism by cytochrome P450 was implemented,
which helped in proposing the metabolic pathway of ravidasvir. The most dominant
metabolite in rat liver microsomal samples was oxidative ravidasvir, where one
O-demethylated metabolite and eight isomers of the oxidative ravidasvir metabolites
were identified. The study provides essential data for proposing the metabolic
pathway and successfully applied it to determine the pharmacokinetics of ravidasvir
in rat plasma.

KEYWORDS
drug metabolism, hepatitis C virus, LC–MS/MS, linear ion trap, pharmacokinetics, ravidasvir

1 | I N T RO DU CT I O N Some new drug classes targeting the various hepatitis C replica-

tion cycle stages were examined in the past decade. The release and
Hepatitis C virus (HCV) infection is considered as a global challenge development of HCV NS5A inhibitors taken once daily with antici-
health problem. Liver diseases are mainly associated with HCV infec- pated antiviral activity and safety profiles have been a dominant
tion and affect about 2.8% of the human population (Messina driver of the pharmaceutical industry over the past few years
et al., 2015). HCV infection has been regarded as one of the leading (Macdonald & Harris, 2004). Several NS5A inhibitors have demon-
causes of chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, strated clinical utility and have been approved for marketing in multi-
and end-stage liver disease (Martins et al., 2011). ple countries (Bachofner et al., 2018; Gitto et al., 2017). Among

Biomedical Chromatography. 2021;35:e5146. wileyonlinelibrary.com/journal/bmc © 2021 John Wiley & Sons, Ltd. 1 of 14
https://doi.org/10.1002/bmc.5146
 Talanta 238 (2022) 123009

Contents lists available at ScienceDirect

Talanta
journal homepage: www.elsevier.com/locate/talanta

Selective sensing of the nucleoside analogue, trifluridine and tipiracil in

dosage form and biological matrices
Wael Talaat a, *, Mohamed M.Y. Kaddah b, Reda Mohammed Keshk c
a
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt
b
Pharmaceutical and Fermentation Industries Development Center, City of Scientific Research and Technological Applications, New Borg El-Arab, 21934, Alexandria,
Egypt
c
Department of Chemistry, Faculty of Science, Damanhour University, Damanhour, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: A new fluorescent sensor is introduced to analyze nucleoside analogue, trifluridine and tipiracil in tablets and
Quenching biological fluids. The synthesized fluorophore exhibits good fluorescence at 446 nm after excitation at 257 nm.
Trifluridine The interaction between the studied drugs and the reagent was a quenching effect. Different experimental pa­
Tipiracil dosage form
rameters and the mechanism of quenching were discussed. The present method was utilized to analyze tri­
Biological fluids
Furopyridine
fluridine and tipiracil raw materials and tablets over the concentration range of 20–1000 ng/mL and spiked
biological fluids over the range of 30–1000 ng/mL. The method is selective, specific, and possesses good accuracy
and high precision. The method is highly sensitive, with detection limits of 5.8 and 6.0 ng/mL for trifluridine and
tipiracil, respectively, and quantitation limits of 17.7 and 18.1 ng/mL for trifluridine and tipiracil, respectively.
In vivo analysis of trifluridine was achieved selectively and the mean pharmacokinetic parameters were studied.

1. Introduction plasma levels of trifluridine by 22 fold [2]. In addition to the use of

trifluridine as a chemotherapeutic agent, it is used as an anti-herpes
Nucleoside analogues are chemical compounds with structural sim­ virus to treat keratitis and keratoconjunctivitis [1]. The noticed
ilarity to nucleosides. They contain two parts, nucleic acid analogue adverse effects of trifluridine were gastrointestinal symptoms (nausea,
part, which is substituted pyrimidine or purine bases, and sugar parts. vomiting, diarrhea) but they are very rare [2] and neutropenia. A clin­
Due to their unique structure, they can serve as antiviral and anticancer ical study was performed to evaluate the onset of grade≥3 neutropenia
agents. The nucleoside analogue undergoes in vivo phosphorylation and during the first-cycle in patients with metastatic colorectal cancer
becomes very similar to nucleotide and, hence, incorporated into viral treated with trifluridine/tipiracil and its relationship with clinical pa­
DNA replication. The substituent on the nucleic acid analogue prevents rameters of interest, such as overall survival (OS). Trifluridine/tipiracil
base-pairing, resulting in inhibiting the DNA replication process [1]. was used in third-line treatment in 10 patients. The study revealed that
Trifluridine is a deoxyuridine analogue with a chemical structure shown neutropenia is about 40% and is not of clinical significance where the
in (Fig. 1). It is 1-[4-Hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-(tri­ onset of grade ≥3 neutropenia at the first-cycle is associated with longer
fluoromethyl)-(1H,3H)-pyrimidine-2,4-dione [1]. It undergoes phos­ median OS [3]. Due to the importance of trifluridine in cancer treat­
phorylation with thymidine kinase to be incorporated in the DNA ment, a simple probe is needed for monitoring its concentration in pa­
replication. The trifluoromethyl subsitituent inhibits base pairing and tient plasma in particular in relation to the neutropenia and indirectly
hence inhibit the process of DNA replication [2]. Trifluridine is used in with the possible correlation with OS.
combination with tipiracil for the treatment of cancer. Tipiracil is a Trifluridine was analyzed either alone or in combination with tipir­
pyrimidine analogue (5-Chloro-6-[(2-imino-1-pyrrolidinyl)methyl]-2,4 acil by several reported methods. The United States Pharmacopoeia
(1H,3H)-pyrimidinedione) (Fig. 1). Tipiracil inhibits trifluridine meta­ reported high performance chromatographic (HPLC) method for its
bolism through inhibition of thymidine phosphorylase, which degrades determination [4]. Other chromatographic methods were also reported
it. Hence, trifluridine and tipiracil’s co-formulation results in increased [5–8] in addition to a capillary electrophoretic [9] and

* Corresponding author.
E-mail addresses: wtalaat2017@gmail.com, waeltalaat@pharm.dmu.edu.eg (W. Talaat), mmkaddah1978@gmail.com (M.M.Y. Kaddah), keshk_reda@yahoo.com
(R.M. Keshk).

https://doi.org/10.1016/j.talanta.2021.123009
Received 26 May 2021; Received in revised form 22 October 2021; Accepted 27 October 2021
Available online 28 October 2021
0039-9140/© 2021 Elsevier B.V. All rights reserved.
 RSC Advances
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.

View Article Online

PAPER View Journal | View Issue

Unravelling the antifungal and antiprotozoal

Cite this: RSC Adv., 2022, 12, 2980
activities and LC-MS/MS quantification of steroidal
Open Access Article. Published on 21 January 2022. Downloaded on 7/19/2022 3:39:43 PM.

saponins isolated from Panicum turgidum†

Ahmed A. Zaki, *ab Mohamed M. Y. Kaddah, c
Hamada S. Abulkhair *de
and Ahmed Ashour ab

Bioassay-guided investigation of Panicum turgidum extract resulted in the identification of seven steroidal
saponins (Turgidosterones 1–7). They were evaluated for their in vitro antifungal, antileishmanial, and
antitrypanosomal activities. Turgidosterone 6 was the most active antifungal against Candida albicans
and Candida neoformans (IC50 values of 2.84 and 1.08 mg mL
1, respectively). Turgidosterones 4–7
displayed antileishmanial activity against Leishmania donovani promastigotes with IC50 values ranging
from 4.95 to 8.03 mg mL
1 and against Leishmania donovani amastigote/THP with IC50 values range of
4.50–9.29 mg mL
1. Activity against Trypanosoma brucei was also observed for Turgidosterones 4–7
with an IC50 values range of 1.26–3.77 mg mL
1. Turgidosterones 1–3 did not display any activity against
the tested pathogens. The study of structure–activity relationships of the isolated saponins indicated that
the antifungal, antileishmanial, and antitrypanosomal activities are markedly affected by the presence of
spirostane-type saponins and the elongation of the sugar residue at C-3. To quantitatively determine the
most abundant active ingredient in Panicum turgidum extract, a single run, sensitive, and highly selective
liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been applied under positive
and negative modes. The obtained results showed that compound 5 was the most abundant (95.93 
Received 21st November 2021
Accepted 15th January 2022
1.10 mg per gram of dry Panicum turgidum extract), followed by 6 (52.51  1.05 mg gm
1), 4 (32.71 
0.48 mg gm
1), and 7 (13.19  0.50 mg gm
1). Docking of these saponins against the Candida albicans
DOI: 10.1039/d1ra08532h
oxidoreductases and Leishmania infantum trypanothione reductase active sites revealed their potential
rsc.li/rsc-advances to effectively bind with a number of key residues in both receptor targets.

hypercholesterolemia, cardiovascular disease, liver injury, and

1. Introduction type II diabetes.5,6 Panicum turgidum Forssk. is a widely
Panicum (panic grass) is one of the important genera belonging distributed grass in the Egyptian desert. In previous work, we
to the family Poaceae.1 Traditionally, it has been reported to have reported the anti-inammatory activity of extracts and
treat various ailments. The genus Panicum includes 450 species isolated compounds from P. turgidum.1,7 Moreover, in our recent
native to the tropical regions, with a few species extending into publication, the isolation of steroidal saponins and their anti-
the northern temperate zone.2 P. maximum was reported to have proliferative activity was studied.5 However, the antifungal and
antimalarial, antirheumatic, anti-inammatory, antidiabetic, antiprotozoal activities of P. turgidum have not yet been
antiplasmodial, and analgesic activities.3,4 It was reported that investigated.
the consumption of P. miliaceum grains protects against Microbes and protozoa are the leading causes of infectious
diseases, death, and many global health issues.8–10 The treat-
a
ment of most pathogens is difficult because of their resistance
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura
to traditional medications. Additionally, antifungal drugs such
35516, Egypt. E-mail: ahmed.awad@fulbrightmail.org
b
Department of Pharmacognosy, Faculty of Pharmacy, Horus University-Egypt,
as amphotericin B which is effective against candida infections
International Coastal Road, New Damietta 34518, Egypt showed severe side effects, including renal toxicity.11 Therefore,
c
Pharmaceutical and Fermentation Industries Development Center, City of Scientic there is an urgent unmet need to develop new safe antifungal
Research and Technological Applications, New Borg El-Arab 21934, Alexandria, Egypt agents. On one hand, antimony (pentavalent antimonials) is the
d
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar most effective treatment for Leishmania infections.12 Alterna-
University, Nasr City 11884, Cairo, Egypt. E-mail: hamadaorganic@azhar.edu.eg
e
tive drugs commonly used for leishmaniasis are amphotericin B
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Horus University-Egypt,
and pentamidine, which are also considered ineffective.13 On
International Coastal Road, New Damietta 34518, Egypt
† Electronic supplementary information (ESI) available. See DOI:
the other hand, nifurtimox and benznidazole are the front-line
10.1039/d1ra08532h antitrypanosomal against acute Chagas' disease.14 They are

2980 | RSC Adv., 2022, 12, 2980–2991 © 2022 The Author(s). Published by the Royal Society of Chemistry
 Biomedicine & Pharmacotherapy 148 (2022) 112731

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Nifuroxazide-loaded cubosomes exhibit an advancement in pulmonary

delivery and attenuate bleomycin-induced lung fibrosis by regulating the
STAT3 and NF-κB signaling: A new challenge for unmet therapeutic needs
Sameh Saber a, *, Mohamed Nasr b, c, **, Mohamed M.Y. Kaddah d, Gomaa Mostafa-Hedeab e, f,
Simona Cavalu g, Ahmed A.E. Mourad h, Ahmed Gaafar Ahmed Gaafar h, Sameh S. Zaghlool i,
Safaa Saleh j, Mohamed M. Hafez k, Samuel Girgis l, Rehab Mohamed Elgharabawy m,
Karim Nader n, Mansour Alsharidah o, Gaber El-Saber Batiha p, Eman El-Ahwany q,
◦

Noha A. Amin r, Heba I. Elagamy c, Ahmed Shata s, t, Reem Nader n, Ahmed E. Khodir u
a
Department of Pharmacology, Faculty of Pharmacy, Delta University for Science and Technology, Gamasa 11152, Egypt
b
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Helwan University, Cairo 11790, Egypt
c
Department of Pharmaceutics, Faculty of Pharmacy, Delta University for Science and Technology, Gamasa, Egypt
d
Pharmaceutical and Fermentation Industries Development Center, City of Scientific Research and Technological Applications, New Borg El-Arab 21934, Alexandria,
Egypt
e
Pharmacology Department & Health Research Unit, Medical College, Jouf University, Saudi Arabia
f
Pharmacology Department, Faculty of Medicine, Beni-Suef University, Beni Suef, Egypt
g
Faculty of Medicine and Pharmacy, University of Oradea, P-ta 1 Decembrie 10, 410087 Oradea, Romania
h
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Port Said University, Port Said 42511, Egypt
i
Pharmacology and Toxicology Department, Faculty of Pharmacy, Modern University for Technology and Information (MTI), Mokattam, Cairo 11571, Egypt
j
Department of Clinical Physiology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
k
Department of Biochemistry, Faculty of Pharmacy, Ahram Canadian University, Giza, Egypt
l
Department of Pharmaceutics, Faculty of Pharmacy, Alsalam University, Egypt
m
Pharmacology and Toxicology Department, Faculty of Pharmacy, Tanta University, Tanta, Egypt
n
Department of Biochemistry, Faculty of Pharmacy, Delta University for Science and Technology, Gamasa 11152, Egypt
o
Department of Physiology, College of Medicine, Qassim University, Qassim 51452, Kingdom of Saudi Arabia
p
Department of Pharmacology and Therapeutics, Faculty of Veterinary Medicine, Damanhour University, Damanhour 22511, AlBeheira, Egypt
q
Department of Immunology, Theodor Bilharz Research Institute, Giza 12411, Egypt
r
Department of Haematology, Theodor Bilharz Research Institute, Giza 12411, Egypt
s
Department of Clinical Pharmacology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
t
Department of Clinical Pharmacy, Faculty of Pharmacy, Delta University for Science and Technology, Gamasa, Egypt
u
Department of Pharmacology, Faculty of Pharmacy, Horus University, New Damietta, Egypt

Abbreviations: BLMC, bleomycin; COL1A1, Collagen, type I alpha 1; ECM, extracellular matrix; EE, entrapment efficiency; GMO, glyceryl monooleate; GSH,
reduced glutathione; IPF, idiopathic pulmonary fibrosis; ICAM-1, intercellular adhesion molecule-1; ITBM, intratracheal bleomycin model; MCP-1, monocyte che­
moattractant Protein-1; MDA, malondialdehyde; NF-κB, nuclear factor kappa B; PDGF-BB, platelet derived growth factor-BB; NXZD, nifuroxazide; PF, pulmonary
fibrosis; SOD, superoxide dismutase; STAT3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor beta; TIMP-1, metalloproteinase-1;
TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor alpha.
* Correspondence to: Department of Pharmacology, Faculty of Pharmacy, Delta University for Science and Technology, Costal International Road, Gamasa,
Dakahlia, Egypt.
** Corresponding author at: Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Helwan University, Cairo 11790, Egypt.
E-mail addresses: sampharm81@gmail.com, sameh.mohamed@deltauniv.edu.eg (S. Saber), m2nasr@yahoo.com (M. Nasr), mmkaddah1978@gmail.com
(M.M.Y. Kaddah), gomaa@ju.edu.sa (G. Mostafa-Hedeab), simona.cavalu@gmail.com (S. Cavalu), ahmed.mourad@yahoo.com (A.A.E. Mourad), agafer777@
yahoo.com (A.G.A. Gaafar), samehsaadzaghlool@yahoo.com, sameh.s.zaghlool@pharm.mti.edu.eg (S.S. Zaghlool), safaa_kotb48@yahoo.com (S. Saleh),
mohkhalifa2000@yahoo.com (M.M. Hafez), girgissamuel2@gmail.com (S. Girgis), drrehab200932@yahoo.com (R.M. Elgharabawy), kareemnadersamra@gmail.
com (K. Nader), malsharidah@qu.edu.sa (M. Alsharidah), gaberbatiha@gmail.com (G.E.-S. Batiha), ahwany@aucegypt.edu (E. El-Ahwany), esmaealn78@gmail.
com (N.A. Amin), hebaelagamy1985@yahoo.com (H.I. Elagamy), ahmedmhes@mans.edu.eg (A. Shata), reemnadersamra@gmail.com (R. Nader), Dr.ahmed_
esam@yahoo.com (A.E. Khodir).

https://doi.org/10.1016/j.biopha.2022.112731
Received 27 December 2021; Received in revised form 10 February 2022; Accepted 15 February 2022
Available online 24 February 2022
0753-3322/© 2022 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
 www.nature.com/scientificreports

OPEN A sustainable and effective

bioprocessing approach
for improvement of acid
phosphatase production and rock
phosphate solubilization
by Bacillus haynesii strain ACP1
Soad A. Abdelgalil1*, Mohamed M. Y. Kaddah2, Mahmoud E. A. Duab1 & Gaber A. Abo‑Zaid1

There is indeed a tremendous increase in biotechnological production on a global scale, more and
more innovative bioprocesses, therefore, require to perform ideally not only in a small lab- but also
on large production scales. Efficient microbial process optimization is a significant challenge when
accomplishing a variety of sustainable development and bioengineering application objectives. In
Egypt’s mines, several distinct types of rock phosphate (RP) are utilized as a source of phosphate
fertilizers in agriculture. It is more ecologically beneficial to utilize RP bio-solubilization than
acidulation. Therefore, this work aimed to strategically scale up the acid phosphatase (ACP)
production and RP bio-solubilization by the newly-discovered Bacillus haynesii. The use of consecutive
statistical experimental approaches of Plackett–Burman Design (PBD), and Rotatable Central
Composite Design (RCCD), followed by pH-uncontrolled cultivation conditions in a 7 L bench-top
bioreactor revealed an innovative medium formulation. These approaches substantially improved
ACP production, reaching 207.6 U ­L−1 with an ACP yield coefficient Yp/x of 25.2 and a specific growth
rate (µ) of 0.07 ­h−1. The metals Na, Li, and Mn were the most efficiently released from RP during
the solubilization process by B. haynesii. The uncontrolled pH culture condition is the most suitable
setting for simultaneously improving the ACP and organic acids production. The most abundant
organic acid produced through the cultivation process was lactic acid, followed by glutamic acid and
hydroxybenzoic acid isomer. The findings of TGA, DSC, SEM, EDS, FTIR, and XRD analysis emphasize
the significant influence of organic acids and ACP activity on the solubilization of RP particles.

Today’s globalization has forced sustainable agriculture to fulfill our agricultural requirements, which conven-
tional agriculture cannot. Agricultural production systems must be intensified to support productivity gains and
revenue generation to maintain food security in developing countries. Among the necessary macronutrients for
higher plants and animals is phosphorus (P), whereas, numerous metabolic pathways require the presence of P
to operate. Egypt has alkali soils that make phosphate minerals exceedingly challenging to dissolve. Using RP as
a source of P in the right way can contribute to sustainable agricultural intensification, especially in developing
countries with RP d­ eposits1. In mineralogy and geology, phosphates are rocks or ore that contain phosphate ions.
Phosphorite, also known as RP, is an exhaustible nonrenewable natural resource of a non-detrital sedimentary
rock that includes substantial quantities of phosphate-bearing m ­ inerals2. Different geological rocks may be
found across the world.

1
Bioprocess Development Department, Genetic Engineering, and Biotechnology Research Institute (GEBRI),
City of Scientific Research and Technological Applications, New Borg El‑Arab City, Alexandria 21934,
Egypt. 2Pharmaceutical and Fermentation Industries Development Center, City of Scientific Research and
Technological Applications, New Borg El‑Arab City, Alexandria 21934, Egypt. *email: Soad_abu_bakr@
yahoo.com

Scientific Reports | (2022) 12:8926 | https://doi.org/10.1038/s41598-022-11448-6 1

Vol.:(0123456789)
Received: 25 April 2022 Revised: 7 July 2022 Accepted: 26 July 2022
DOI: 10.1002/bmc.5472

RESEARCH ARTICLE

New quantification method for monitoring 18 L-amino acids

levels in schizophrenic patients by high-performance liquid
chromatography coupled to tandem quadrupole mass
spectrometer

Mohamed M. Y. Kaddah1 | Heba M. Ali2 | Sherin F. Hammad2 | Samah F. El-Malla2

1
Pharmaceutical and Fermentation Industries
Development Center, City of Scientific Abstract
Research and Technological Applications, New
A fast, uncomplicated, sensitive and fully validated high-performance liquid
Borg El-Arab, Alexandria, Egypt
2
Department of Pharmaceutical Analytical
chromatography–tandem mass spectrometry (HPLC–MS/MS) method has been
Chemistry, Faculty of Pharmacy, Tanta developed for estimating L-amino acids in the plasma of schizophrenic patients. The
University, Tanta, Egypt
gradient-elution chromatographic method was implemented with the Luna® PFP col-
Correspondence umn (50  2.0 mm, 5 μm), and a mobile phase of 0.1% formic acid in water and meth-
Mohamed Mohamed Yousri Kaddah, City of
Scientific Research and Technological
anol was used. The intra- and interday variability of the L-amino acids was <13.11%,
Applications Pharmaceutical and Fermentation their accuracy ranged from 85.14 to 116.75% at the quality control levels and the
Industries Development Center New Borg
El-Arab 21934, Alexandria Egypt. lower limit of quantification ranged from 2.5 to 15 nM. The extraction efficiency
Email: mmkaddah1978@gmail.com and (apparent recovery) of amino acids from healthy plasma was employed by spiking the
mkaddah@srtacity.sci.eg
plasma with standard amino acids at the quality control levels. Their percentage
recoveries ranged from 80.4 to 119.94%. Our method has a short run time and fast
sample preparation compared with existing methods, which suffer from long prepara-
tive steps and/or time-consuming analysis, restricted reagents and the suboptimal
performance characteristics of presently available technologies. Therefore, the pro-
posed HPLC–MS/MS method was effectively applied for monitoring the L-amino
acids in the plasma of schizophrenic patients and healthy volunteers.

KEYWORDS
amino acids, liquid chromatography–tandem mass spectrometry, metabolic disorders,
schizophrenia

1 | I N T RO DU CT I O N cannot produce 10 essential amino acids among all of the endogenous

amino acids and these must be provided through food (Tymoczko
Amino acids (AAs) are the building units of proteins. All amino acids et al., 2011). Amino acids play crucial roles as intermediates in metab-
have the same basic structure that consists of an alpha (α) central or olism, neurotransmission and the building blocks of proteins (Chae
chiral carbon atom. A carboxyl group (-COOH), an amino group et al., 2020; Wu, 2009). Some AAs such as glutamate, aspartate and
(-NH2), a hydrogen atom and a specific R-group are connected to an α glycine are the most abundant neurotransmitters in the brain (He &
carbon atom. According to the specific R group attached to the α car- Wu, 2020). Estimation of free amino acids in physiological fluids is
bon atom, 20 amino acids are found in proteins (Tymoczko crucial to the biomarkers of a wide variety of central nervous system
et al., 2011). The side chain (R) is responsible for the chemical proper- diseases and estimation of nutritional status, dietary compliance, renal
ties of amino acids, so they are classified into four groups—polar, non- function and tissue damage (Dalangin et al., 2020; Khalil et al., 2018;
polar, aromatic and branched-chain amino acids. The human body Prado et al., 2020; Tizianello et al., 1980). Furthermore, several

Biomedical Chromatography. 2022;e5472. wileyonlinelibrary.com/journal/bmc © 2022 John Wiley & Sons Ltd. 1 of 17
https://doi.org/10.1002/bmc.5472
Received: 19 June 2022 Revised: 19 August 2022 Accepted: 8 September 2022

DOI: 10.1002/jssc.202200481

RESEARCH ARTICLE

Quantification of 16 cephalosporins in the aquatic

environment by liquid chromatography-tandem mass
spectrometry

Mohamed M. Y. Kaddah1 Emad H. Al-Dokhmaisy2 Basem Mansour3

Hoda G. Daabees2 Miranda F. Kamal2

1 Pharmaceutical and Fermentation

Industries Development Center, City of Antimicrobial agents are essential to protect human and animal health. During
Scientific Research and Technology the coronavirus disease 2019 pandemic, antimicrobials such as cephalosporins
Applications, Alexandria, Egypt
2 Department
were widely used as prophylactics and to prevent bacterial co-infection.
of Pharmaceutical
Analytical Chemistry, Faculty of Undoubtedly, the prevalence of antibiotics in the aquatic environment will ulti-
Pharmacy, Damanhour University, mately affect the degree of resistance against these bacteria in animals and the
Damanhour, Egypt
environmental systems. In order to monitor 16 cephalosporins in the aquatic
3 Department of Pharmaceutical
environment, we developed a new liquid chromatography-tandem mass spec-
Chemistry, Faculty of Pharmacy, Delta
University for Science and Technology, trometry method that functioned simultaneously under positive and negative
Dakahlia, Egypt electrospray ionization switching modes. The chromatographic separation has
Correspondence
been implemented using a pentafluorophenyl propyl column kept at 40◦ C.
Mohamed Mohamed Yousri Kaddah, City The limits of detection and quantitation for the studied cephalosporins ranged
of Scientific Research and Technological from (8 × 10−4 ) to (7.11 × 10−2 ) ng/ml and from (2.61 × 10−3 ) to (2.37 ×
Applications, Pharmaceutical and
Fermentation Industries Development 10−1 ) ng/ml, respectively. The percent extraction efficiency (apparent recov-
Center, New Borg El-Arab 21934, ery) and relative standard deviations for the analyzed cephalosporins ranged
Alexandria, Egypt.
from 61.69% to 167.67% and 2.45% to 13.48%, respectively. The overall findings
Email: mmkaddah1978@gmail.com,
mkaddah@srtacity.sci.eg showed that the effluent from the wastewater treatment plants that receive
wastewater from pharmaceutical factories had a higher detected amount of
Funding information
cephalosporins than that of domestic sewage. Moreover, seven cephalosporins,
Science, Technology, and Innovation
Funding Authority including cefuroxime, ceftazidime, cefradine, cefprozil, cefixime, cefalexin, and
cefadroxil (0.68–105.45 ng/L) were determined in the aquatic environment.

KEYWORDS
aquatic environment, cephalosporins, liquid chromatography-tandem mass spectrometry,
solid-phase extraction

Article Related Abbreviations: CFAC, cefaclor; CFAD, cefadroxil; CFAZ, cefazolin; CFIX, cefixime; CFOT, cefotaxime; CFOX, cefoxitine; CFPO-P,
cefpodoxime proxetil; CFPR, cefprozil; CFRA, cefradine; CFTA, ceftazidime; CFTR, ceftriaxone; CFUR, cefuroxime; CFUR-X, cefuroxime axetil;
CPHA, cephalexin; CV, coefficient of variation; HQC, high quality control; IS, internal standard; LQC, low quality control; MQC, medium quality
control; MRM, multiple reaction monitoring; WWTPs, wastewater treatment plants.

J Sep Sci 2022;1–18. www.jss-journal.com © 2022 Wiley-VCH GmbH. 1