Acknowledgements

This work was supported by funding from Agriculture and Agri-Food Canada and Ducks Unlimited Canada under a project of the Watershed Evaluation of Beneficial Management Practices and by a Natural Sciences and Engineering Research Council Collaborative Research and Development grant entitled "Development of an advanced growth and yield model for multipurpose sustainable forest management". Zhengyong Zhao was supported by an NSERC Alexander Graham Bell Canada Graduate Scholarship.


**Aqueous-Phase Production of Secondary Organic Aerosols from Oxidation of Dibenzothiophene (DBT)**

#### **Yu Liu 1 , Junchen Lu 1 , Yanfang Chen 1 , Yue Liu 1 , Zhaolian Ye 2 and Xinlei Ge 1,\***

Received: 13 January 2020; Accepted: 29 January 2020; Published: 30 January 2020

**Abstract:** Intermediate-volatility organic compounds (IVOCs) have been recognized as an important contributor to the secondary organic aerosol (SOA) formation via gas-phase reactions. However, it is unclear whether or not IVOCs-SOA can be produced in the aqueous phase. This work investigated aqueous oxidation of one model compound of IVOCs, dibenzothiophene (DBT). Results show that DBT can be degraded by both hydroxyl radical and the triplet excited states of organic light chromophores ( 3C\*). Aqueous dark oxidation of DBT was also possible. SOA yields of 32% and 15% were found for hydroxyl radical (OH)-mediated photo-oxidation and dark oxidation, respectively. A continuous and significant increase of oxidation degree of SOA was observed during OH photo-oxidation, but not during the dark oxidation. Factor analyses revealed that there was a persistent production of highly oxygenated compounds from the less oxygenated species. OH-initiated photochemical reactions can also produce species with a relatively large light-absorbing ability, while such photo-enhancement due to direct light irradiation and 3C\*-initiated oxidation could occur, but is much less important. In the future, studies on the second-order rate constants, molecular characterization of the oxidation products from this and other IVOCs precursors are needed to better understand the role of this reaction pathway in SOA budget, air quality and climate change.

**Keywords:** dibenzothiophene; intermediate-volatility organic compounds; secondary organic aerosol; aqueous phase; brown carbon


Conduct the precursor ion scans by coupled with the IDA function for automatic performing collision on the two highest intensity MS peaks in the targeting regions of HPLC (Step 3). (Note: IDA is a build-in function of API 4000 QTrap (AB Sciex) for conducting an automatic collision on the highest intensity peak(s) scan.)

In addition to the methods mentioned above, i.e., kinetic study and difference of mass-to-charge (∆m/z) between experimental and nominal results, three other evaluation methods to verify the reliability of the identification results are available: including verification by real samples, by stable isotope distribution patterns, and

Investigation results of unknown degradation product(s) must be verified by the "real samples", i.e., commercial batches or long-term/accelerated stability studies samples. Verification of reliability is achieved by comparison the difference of retention time (tR), MRM pairs, and stable isotope distribution patterns between real samples and stress study samples. If it is available, purified or enrichment sample of impurity can be spiked into a real sample for further

2.Verification by stable isotope distribution patterns or natural abundances

the algorithms of **Figure 5**. Core fragment of m/z 243 was found in the MS/MS study of API. In the meantime, four potential extending core fragments, i.e., m/z 183, m/z 185, m/z 197, and m/z 199 were obtained by the MS/MS studies of interme-

2.Step 2: Predict a set of potential/extending MRM pairs in line with the list obtained in step 1 and then coupled it with the relevant (bio-) transformations under the storage conditions of APIs/drug products for conducting MS/MS

*DOI: http://dx.doi.org/10.5772/intechopen.83849*

scans.

step 3.

established (Step 2).

by mass balance.

1.Verification by real samples

unknown component(s) is proposed.

core fragments established in step 2.

diate and degradation product (Step 1).

**4.3 Verification of degradation products (step 4)**

Steps for the determination of impurities related to degradation of API are illustrated as follows:

*Quality Management and Quality Control - New Trends and Developments*

Linear relationship within dynamic ranges for the quantitation of MRM pairs, i.e., correlation coefficients (r = 1) between precursor ions and product ions is another indication to verify high stability and reproducibility of fragmentation in

*Structures of (a) ethyl cysteinate dimer (ECD), (b) intramolecular disulfide (S-S) product of ECD, i.e., ECDS-S, and (c) intermolecular dimer of ECD and reducing agent (SnCl2), i.e., Sn(ECD)2 (DP#4) [25].*

*Kinetic study of impurities formation by conducting stress studies to distinguish DRIs and PRIs.*

Before using the MRM pairs for impurity scanning, interference of fragments generated from background, matrix, or contaminants such as plasticizers present in the solvents and mobile phase must be verified. Plasticizers, e.g., di(2-ethylhexyl) phthalate (DEHP) are one of the most common contaminants in organic solvents,

Repeat the same procedures mentioned above in **Figure 5** to obtain a comprehensive information of fragments for any available intermediates and degradation products which are received from synthetic division, from contract manufacturing organization (CMO), from a stress study, or stability study sample conducted by the

Steps for the determination of impurities related to degradation of API are

CID conditions of tandem MS [24, 25].

including acetonitrile and alcohol [41].

**124**

R&D team.

**Figure 6.**

**Figure 7.**

illustrated as follows:

One preliminary study was illustrated as shown in **Figure 8** can be used to detail the algorithms of **Figure 5**. Core fragment of m/z 243 was found in the MS/MS study of API. In the meantime, four potential extending core fragments, i.e., m/z 183, m/z 185, m/z 197, and m/z 199 were obtained by the MS/MS studies of intermediate and degradation product (Step 1).

A total of five potential core fragments, coupled with the experience accumulated by degradation products that may be produced by similar chemical structures and prediction of relevant (bio-) transformations reactions under storage conditions, such as oxidation (+O, +2O), dehydration (−H2O, −2(H2O)), remove of carbon dioxide, and remove of acetic acid, a set of MRM pairs for scanning is established (Step 2).

Conduct the precursor ion scans by coupled with the IDA function for automatic performing collision on the two highest intensity MS peaks in the targeting regions of HPLC (Step 3). (Note: IDA is a build-in function of API 4000 QTrap (AB Sciex) for conducting an automatic collision on the highest intensity peak(s) scan.)

#### **4.3 Verification of degradation products (step 4)**

In addition to the methods mentioned above, i.e., kinetic study and difference of mass-to-charge (∆m/z) between experimental and nominal results, three other evaluation methods to verify the reliability of the identification results are available: including verification by real samples, by stable isotope distribution patterns, and by mass balance.

1.Verification by real samples

Investigation results of unknown degradation product(s) must be verified by the "real samples", i.e., commercial batches or long-term/accelerated stability studies samples. Verification of reliability is achieved by comparison the difference of retention time (tR), MRM pairs, and stable isotope distribution patterns between real samples and stress study samples. If it is available, purified or enrichment sample of impurity can be spiked into a real sample for further verification.

2.Verification by stable isotope distribution patterns or natural abundances

#### **Figure 8.**

*Step for the establishment of potential extending core fragments, conduct of product ions screening with transformation/IDA function, and validation/verification.*

Each element, like a fingerprint, has its own unique stable isotope distribution patterns and natural abundances. Occasionally, stable isotope distribution patterns or natural abundances are available as a unique tool for structure characterization.

Ten, two, and two of uncommon patterns in the MS spectra as shown in **Figure 9(a)**–**(c)** were clearly indicated in our structure identification of ethyl cysteinate dimer (ECD) cold kit, (R)-N-methyl-3-(2-bromophenoxy)-3 phenylpropanamine (MBPP), and methoxyisobutylisonitrile (sestamibi, or Cu(MIBI)4), respectively. These uncommon patterns were attributed to the contribution of stable isotope distributions of tin (Sn), bromine (Br), and copper (Cu), respectively.

When 7 major (or actually total 10) peaks are shown in the MS spectra, it may strongly mislead the works of structure elucidation as shown in **Figure 9(a)**. However, if it is available to know the presence of some special elements may present in impurity.

If it is able to presuppose that some special elements may contain in the structure, then it will be easier to elucidate the MS spectra. In other words, when pattern of MS spectra is significantly different from the normal CHO distribution, it may also indicate that a special element exists on the structure.

By comparing the natural abundance of 10 stable isotopes of tin and simulation MS spectra of a promising molecular formula, a series of metal complexes of tin can be verified. In the case for study of impurities in ECD kit, it was an ultimate and effective way to identify all of impurities containing Sn, i.e., DP#4, DP#5, DP#6, DP#6′, DP#6″, DP#7, DP#7′, DP#7″, and DP#8 [25]. Similar case was found in

**127**

**Figure 9.**

3.Verification by mass balance

*Determination of Impurities in Pharmaceuticals: Why and How?*

the structure determination of sestamibi as shown in **Figure 9(c)**. Coordination number (CN = 4) and core metal (Cu) in sestamibi can be clearly verified.

*Stable isotope distribution patterns and simulation of mass spectra of (a) Sn(ECD)2 (DP#4), (b) (R)-Nmethyl-3-(2-bromophenoxy)-3-phenylpropanamine (MBPP), and (c) methoxyisobutylisonitrile (sestamibi).*

When performing a stress study of API, one should determine content of API on each day by using a daily and freshly prepared calibration curve of API reference material, and interpolated within the validated dynamic range. The mass balance is

*DOI: http://dx.doi.org/10.5772/intechopen.83849*

*Determination of Impurities in Pharmaceuticals: Why and How? DOI: http://dx.doi.org/10.5772/intechopen.83849*

*Quality Management and Quality Control - New Trends and Developments*

Each element, like a fingerprint, has its own unique stable isotope distribution patterns and natural abundances. Occasionally, stable isotope distribution patterns or natural abundances are available as a unique tool for structure characterization. Ten, two, and two of uncommon patterns in the MS spectra as shown in **Figure 9(a)**–**(c)** were clearly indicated in our structure identification of ethyl cysteinate dimer (ECD) cold kit, (R)-N-methyl-3-(2-bromophenoxy)-3 phenylpropanamine (MBPP), and methoxyisobutylisonitrile (sestamibi, or Cu(MIBI)4), respectively. These uncommon patterns were attributed to the contribution of stable isotope distributions of tin (Sn), bromine (Br), and cop-

*Step for the establishment of potential extending core fragments, conduct of product ions screening with*

When 7 major (or actually total 10) peaks are shown in the MS spectra, it may

If it is able to presuppose that some special elements may contain in the structure, then it will be easier to elucidate the MS spectra. In other words, when pattern of MS spectra is significantly different from the normal CHO distribution, it may

By comparing the natural abundance of 10 stable isotopes of tin and simulation MS spectra of a promising molecular formula, a series of metal complexes of tin can be verified. In the case for study of impurities in ECD kit, it was an ultimate and effective way to identify all of impurities containing Sn, i.e., DP#4, DP#5, DP#6, DP#6′, DP#6″, DP#7, DP#7′, DP#7″, and DP#8 [25]. Similar case was found in

strongly mislead the works of structure elucidation as shown in **Figure 9(a)**. However, if it is available to know the presence of some special elements may pres-

also indicate that a special element exists on the structure.

**126**

per (Cu), respectively.

*transformation/IDA function, and validation/verification.*

ent in impurity.

**Figure 8.**

#### **Figure 9.**

*Stable isotope distribution patterns and simulation of mass spectra of (a) Sn(ECD)2 (DP#4), (b) (R)-Nmethyl-3-(2-bromophenoxy)-3-phenylpropanamine (MBPP), and (c) methoxyisobutylisonitrile (sestamibi).*

the structure determination of sestamibi as shown in **Figure 9(c)**. Coordination number (CN = 4) and core metal (Cu) in sestamibi can be clearly verified.

#### 3.Verification by mass balance

When performing a stress study of API, one should determine content of API on each day by using a daily and freshly prepared calibration curve of API reference material, and interpolated within the validated dynamic range. The mass balance is

calculated by summation of the API and total impurity content. It is a tool to justify whether there are impurities unseparated (i.e., same retention time) or undetectable (e.g., without UV-visible chromophores). This topic and several major problems to cause poor mass balance have been detailed by Nussbaum et al. [42]

#### **5. Conclusions**

Management of impurities related to APIs in pharmaceutical products must be implemented in strict compliance with the regulatory requirements of pharmaceutical industry due to their quality and safety concerns. An integrated scheme in accordance with the regulatory requirements to establish analytical methods and acceptance criteria of process-related impurities (PRIs) and degradation-related impurities (DRIs) was presented, accordingly. Meanwhile, procedures for the identification and validation/verification of API-related DRIs were proposed. Validation or verification methods to evaluate the reliability of structure identification such as kinetic reactions, stress and stability studies, comparison of retention time(s) and ∆m/z between experimental and nominal values of targeting peaks, compatibility of MRM pairs with "real samples," stable isotope distribution patterns, and mass balance were demonstrated. Applying of the processes proposed in this article will help to ensure the reliability and quality of the impurity analytical results.

#### **Acknowledgements**

The authors would like to thank Dr. Shyh-Fong Chen, Mr. Chu-Huang Hsieh, Miss Jane-Yu Huang, Miss Pi-Lin Liu, and Mr. Yung-Hsuan Sung, Pharmaceutical Business Unit, Everlight Chemical Industrial Corporation (ECIC) for their helpful advice on the Regulatory Requirements in this chapter. The authors also appreciate Dr. Lai-Chuan Chang, Biotech Total Solutions Co., Ltd., Dr. Lee-Chung Men, Dr. Lie-Hang Shen, Dr. Mei-Hsiu Liao, Miss Yi-Chih Hsia, and Mr. Chang-Yung Su, Institute of Nuclear Energy Research (INER) for their great supporting on the radiopharmaceutical and analytical works.

#### **Author details**

Kung-Tien Liu1 \* and Chien-Hsin Chen2

1 Pharmaceutical Business Unit, Everlight Chemical Industrial Corporation (ECIC), Taipei, Taiwan

2 Everlight Chemical Industrial Corporation (ECIC), Taipei, Taiwan

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**129**

2014

2009

*Determination of Impurities in Pharmaceuticals: Why and How?*

of structure activity relationships (SAR) in toxicology. Toxicological Sciences.

[11] Kleinman MH, Elder D, Teasdale A, Mowery MD, McKeown AP, Baertschi SW. Strategies to address mutagenic impurities derived from degradation in drug substances and drug products.

Organic Process Research and Development. DOI: 10.1021/acs.

[12] Wasylaschuk WR, Harmon PA, Wagner G, Harman AB, Templeton AC, Xu H, et al. Evaluation of hydroperoxides in common pharmaceutical excipients. Journal of Pharmaceutical Sciences. 2007;**96**(1):106-116. DOI: 10.1002/

[13] Prachi S, Komal C, Priti MJ. Influence of peroxide impurities in povidone on the stability of selected β-blockers with the help of HPLC. AAPS

PharmSciTech. 2017;**18**(7). DOI: 10.1208/s12249-017-0716-2

[15] Kiehl D. Characterization of Extractables and Leachables Associated

with Pharmaceutically Relevant Materials: Case Studies Outlining Analytical Approaches, Challenges and Examples. Indianapolis, IN, USA: Eli Lilly & Company. http:// apps.thermoscientific.com/media/ SID/LSMS/PDF/LSMSUsersMtg/ Indianapolis/DKiehl\_Thermo\_User\_

[16] Müller L, Mauthe RJ, Riley CM, Andino MM, De Antonis D, Beels C, et al. A rationale for determining, testing, and controlling specific

Meeting\_Seminar.pdf

[14] Fathima N, Mamatha T, Qureshi HK, Anitha N, Rao JV. Drug-excipient interaction and its importance in dosage form development. Journal of Applied Pharmaceutical Science. 2011;**1**(6):66-71

2000;**56**(1):8-17

oprd.5b00091

jps.20726

*DOI: http://dx.doi.org/10.5772/intechopen.83849*

[1] USP Chapters <1086> Impurities in drug substances and drug products. USP 41. The United States Pharmacopeial

Convention. August 1, 2018

2006

**References**

[2] ICH Harmonised Tripartite Guideline. Impurities in new drug substances. Q3A(R2). ICH. 25 October

[3] ICH Harmonised Tripartite Guideline. Impurities in new drug products. Q3B(R2). ICH. 2 June 2006

[4] Crowley P, Martini LG. Drugexcipient interactions. Pharmaceutical

[5] Bharate SS, Bharate SB, Bajaj AN. Interactions and incompatibilities of pharmaceutical excipients with active pharmaceutical ingredients: A comprehensive review. Journal of Excipients and Food Chemicals.

[6] Hotha KK, Roychowdhury S, Subramanian V. Drug-excipient

of drug degradation pathways. American Journal of Analytical Chemistry. 2016;**7**:107-140

[8] ICH Harmonised Tripartite Guideline. Impurities: Guideline for Residual Solvents. Q3C(R6).

[9] ICH Harmonised Tripartite Guideline. Guideline for Elemental Impurities. Q3D. ICH. 16 December

[10] McKinney JD, Richard A, Waller C, Newman MC, Gerberick F. The practice

ICH. October 20, 2016

interactions: Case studies and overview

[7] FDA Guidance. ANDAs: Impurities in Drug Substances. Center for Drug Evaluation and Research (CDER), Food and Drug Administration (FDA); June

Technology. 2001;**13**:26-34

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*Determination of Impurities in Pharmaceuticals: Why and How? DOI: http://dx.doi.org/10.5772/intechopen.83849*

#### **References**

*Quality Management and Quality Control - New Trends and Developments*

calculated by summation of the API and total impurity content. It is a tool to justify whether there are impurities unseparated (i.e., same retention time) or undetectable (e.g., without UV-visible chromophores). This topic and several major problems to cause poor mass balance have been detailed by Nussbaum et al. [42]

Management of impurities related to APIs in pharmaceutical products must be implemented in strict compliance with the regulatory requirements of pharmaceutical industry due to their quality and safety concerns. An integrated scheme in accordance with the regulatory requirements to establish analytical methods and acceptance criteria of process-related impurities (PRIs) and degradation-related impurities (DRIs) was presented, accordingly. Meanwhile, procedures for the identification and validation/verification of API-related DRIs were proposed. Validation or verification methods to evaluate the reliability of structure identification such as kinetic reactions, stress and stability studies, comparison of retention time(s) and ∆m/z between experimental and nominal values of targeting peaks, compatibility of MRM pairs with "real samples," stable isotope distribution patterns, and mass balance were demonstrated. Applying of the processes proposed in this article will

help to ensure the reliability and quality of the impurity analytical results.

The authors would like to thank Dr. Shyh-Fong Chen, Mr. Chu-Huang Hsieh, Miss Jane-Yu Huang, Miss Pi-Lin Liu, and Mr. Yung-Hsuan Sung, Pharmaceutical Business Unit, Everlight Chemical Industrial Corporation (ECIC) for their helpful advice on the Regulatory Requirements in this chapter. The authors also appreciate Dr. Lai-Chuan Chang, Biotech Total Solutions Co., Ltd., Dr. Lee-Chung Men, Dr. Lie-Hang Shen, Dr. Mei-Hsiu Liao, Miss Yi-Chih Hsia, and Mr. Chang-Yung Su, Institute of Nuclear Energy Research (INER) for their great supporting on the

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Pharmaceutical Business Unit, Everlight Chemical Industrial Corporation (ECIC),

2 Everlight Chemical Industrial Corporation (ECIC), Taipei, Taiwan

**128**

**Author details**

**Acknowledgements**

**5. Conclusions**

Kung-Tien Liu1

Taipei, Taiwan

provided the original work is properly cited.

\* and Chien-Hsin Chen2

radiopharmaceutical and analytical works.

\*Address all correspondence to<EMAIL_ADDRESS>
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[4] Crowley P, Martini LG. Drugexcipient interactions. Pharmaceutical Technology. 2001;**13**:26-34

[5] Bharate SS, Bharate SB, Bajaj AN. Interactions and incompatibilities of pharmaceutical excipients with active pharmaceutical ingredients: A comprehensive review. Journal of Excipients and Food Chemicals. 2010;**1**(3):3-26

[6] Hotha KK, Roychowdhury S, Subramanian V. Drug-excipient interactions: Case studies and overview of drug degradation pathways. American Journal of Analytical Chemistry. 2016;**7**:107-140

[7] FDA Guidance. ANDAs: Impurities in Drug Substances. Center for Drug Evaluation and Research (CDER), Food and Drug Administration (FDA); June 2009

[8] ICH Harmonised Tripartite Guideline. Impurities: Guideline for Residual Solvents. Q3C(R6). ICH. October 20, 2016

[9] ICH Harmonised Tripartite Guideline. Guideline for Elemental Impurities. Q3D. ICH. 16 December 2014

[10] McKinney JD, Richard A, Waller C, Newman MC, Gerberick F. The practice of structure activity relationships (SAR) in toxicology. Toxicological Sciences. 2000;**56**(1):8-17

[11] Kleinman MH, Elder D, Teasdale A, Mowery MD, McKeown AP, Baertschi SW. Strategies to address mutagenic impurities derived from degradation in drug substances and drug products. Organic Process Research and Development. DOI: 10.1021/acs. oprd.5b00091

[12] Wasylaschuk WR, Harmon PA, Wagner G, Harman AB, Templeton AC, Xu H, et al. Evaluation of hydroperoxides in common pharmaceutical excipients. Journal of Pharmaceutical Sciences. 2007;**96**(1):106-116. DOI: 10.1002/ jps.20726

[13] Prachi S, Komal C, Priti MJ. Influence of peroxide impurities in povidone on the stability of selected β-blockers with the help of HPLC. AAPS PharmSciTech. 2017;**18**(7). DOI: 10.1208/s12249-017-0716-2

[14] Fathima N, Mamatha T, Qureshi HK, Anitha N, Rao JV. Drug-excipient interaction and its importance in dosage form development. Journal of Applied Pharmaceutical Science. 2011;**1**(6):66-71

[15] Kiehl D. Characterization of Extractables and Leachables Associated with Pharmaceutically Relevant Materials: Case Studies Outlining Analytical Approaches, Challenges and Examples. Indianapolis, IN, USA: Eli Lilly & Company. http:// apps.thermoscientific.com/media/ SID/LSMS/PDF/LSMSUsersMtg/ Indianapolis/DKiehl\_Thermo\_User\_ Meeting\_Seminar.pdf

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[24] Yang HH, Liu KT, Hsia YC, Chen WH, Chen CC, Men LC, et al. Development and validation of an HPLC method for determination of purity of Sn-ADAM, a novel precursor of serotonin transporter SPECT imaging agent I-123-ADAM. Journal of Food and Drug Analysis. 2010;**18**(5):307-318

[25] Liu KT, Lin YY, Hsia YC, Zhao JH, Su CY, Shen SY, et al. Study of degradation products and degradation pathways of ECD and its drug product, ECD kit. In: Akyar I, editor. Wide Spectra of Quality Control. Croatia: InTech; 2011. pp. 105-132. ISBN: 978-953-307-683-6

[26] Alsante KM, Ando A, Brown R, Ensing J, Hatajik TD, Kong W, et al. The role of degradant profiling in active pharmaceutical ingredients and drug products. Advanced Drug Delivery Reviews. 2007;**59**:29-37

[27] Holm R, Elder DP. Analytical advances in pharmaceutical impurity profiling. European Journal of Pharmaceutical Sciences. 2016;**87**:118-135

[28] EMA Guideline. Guideline on the Limits of Genotoxic Impurities. London: Committee for Medicinal Products for Human Use (CHMP), European Medicines Agency; 28 June 2006. EMEA/CHMP/QWP/251344/2006

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Organization; 2014

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QWP/115498/2017

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[31] Committee for Medicinal Products for Human use (CHMP). Implementation Strategy of ICH Q3D Guideline. European Medicines Agency; 08 March 2017. EMA/CHMP/ QWP/115498/2017

*Quality Management and Quality Control - New Trends and Developments*

[24] Yang HH, Liu KT, Hsia YC, Chen WH, Chen CC, Men LC, et al. Development and validation of an HPLC method for determination of purity of Sn-ADAM, a novel precursor of serotonin transporter SPECT imaging agent I-123-ADAM. Journal

of Food and Drug Analysis.

[25] Liu KT, Lin YY, Hsia YC, Zhao JH, Su CY, Shen SY, et al. Study of degradation products and degradation pathways of ECD and its drug product, ECD kit. In: Akyar I, editor. Wide Spectra of Quality Control. Croatia: InTech; 2011. pp. 105-132. ISBN:

[26] Alsante KM, Ando A, Brown R, Ensing J, Hatajik TD, Kong W, et al. The role of degradant profiling in active pharmaceutical ingredients and drug products. Advanced Drug Delivery

[27] Holm R, Elder DP. Analytical advances in pharmaceutical impurity profiling. European Journal of Pharmaceutical Sciences.

[28] EMA Guideline. Guideline on the Limits of Genotoxic Impurities. London: Committee for Medicinal Products for Human Use (CHMP), European Medicines Agency; 28 June 2006. EMEA/CHMP/QWP/251344/2006

[29] Committee for Medicinal Products for Human Use (CHMP). Guideline on the Specification Limits for Residues of Metal Catalysts. London: European Medicines Agency; January 2007. Doc. Ref. CPMP/SWP/QWP/4446/00 corr

[30] Committee for Medicinal Products for Human use (CHMP). Guideline on the Specification Limits for Residues of Metal Catalysts or Metal Reagents. London: European Medicines Agency; 21 February 2008. Doc. Ref. EMEA/

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## *Edited by Paulo Pereira and Sandra Xavier*

Quality management (QM) practices are the basis for the successful implementation and maintenance of any QM system. Quality control (QC) is identified as a QM component. Therefore, QM effectiveness is dependent on the QC strategy. QC practice is more or less complex depending on the type of production. The book is focused on new trends and developments in QM and QC in several types of industries from a worldwide perspective. Its content has been organized into two sections and seven chapters written by well-recognized researchers worldwide. Several approaches are debated based on sample traceability, analytical method validation, required parameters, class of exponential regression-type estimators of the population means, determination of impurities, viewpoints, and case studies.

Published in London, UK © 2019 IntechOpen © industryview / iStock

Quality Management and Quality Control - New Trends and Developments

Quality Management

and Quality Control

New Trends and Developments

*Edited by Paulo Pereira and Sandra Xavier*


**4. The role of SMC-derived foam cells**

Pathological changes in the atherosclerotic intima include increased modification of lipoproteins (e.g., oxidized LDL) and SMC uptake of modified lipoproteins. Foam cell formation by SMCs, in addition to macrophage-derived foam cells, may represent a pivotal Dynamic Interplay Between Smooth Muscle Cells and Macrophages in Vascular Disease http://dx.doi.org/10.5772/61089 237

**3. Existence of immature SMCs in vascular disease**

236 Muscle Cell and Tissue

the repopulation of immature SMC by mature SMC.

is where those intimal SMCs originate from.

**4. The role of SMC-derived foam cells**

SMCs exhibit the diversity of phenotypes in the vascular lesions. A panel of antibodies for SMC differentiation markers enables to identify a wide spectrum of their phenotypes in experimental and human vascular diseases. In the arterial lesions after acute mechanical injury and in chronic atherosclerotic plaques of experimental animals, intimal SMC often express lower levels of SM-MHC isoforms SM1 and SM2, whereas the medial SMC usually stain positively for SM-MHC and α-actin. While SM1 expression appears at the late stage of vascular development, SM2 expression increases later, particularly after birth [2, 35-37]. SM2 thus serves as a sensitive marker of mature SMC. Aikawa et al. demonstrated that intimal SMC of the rabbit aorta 4 months after mechanical injury and the initiation of a high-cholesterol diet showed an immature phenotype as gauged by decreased SM2 immunoreactivity (Figure 1, top panels) [38]. Interestingly, dietary cholesterol lowering for 8 or 16 months induced the increased expression of SM2 in intimal SMC to the levels similar to those of medial SMC (Figure 1, bottom panels). Such SMC plasticity may depend on microenvironmental cues. Alterna‐ tively, different microenvironment may affect the balance of SMC subpopulations. Thus, the recovery of SM2 expression by lipid lowering may result from either "redifferentiation" of the same group of intimal SMC, the expansion of a subset of SMC with a mature phenotype, or

In human coronary arteries, SMC begin to lose their SM2 expression even in the physiological intima of young adults with no obvious signs of atherosclerotic changes such as macrophage accumulation [28]. In more advanced plaques, immature SMCs often exist in areas where macrophages accumulate. In Figure 2, SM2 was undetectable in SMC identified by SMα-actin and SM1. Co-existence of intraplaque microvessels and macrophages may indicate active recruitment of circulating monocytes into this area. A pro-inflammatory microenvironment produced by activated macrophages via the release of IL-1β, PDGF-BB, and other factors and production of oxidative stress may have promoted phenotypic modulation of SMC in this region. As we discuss later, some of these immature SMC may have originated from a subset of monocytes. Alternatively, some SMCs may have transdifferentiated into macrophage-like cells. Understanding of such crosstalk and potential interchangeability between SMCs and macrophages may provide important insight into the identification of new therapeutic targets. Of note, "redifferentiation" or "repopulation" of intimal SMC after mechanical injury also occurs in human coronary arteries. The differentiation state of SMC reduces a first few months after percutaneous coronary intervention while it increases over time [27]. The key question

Pathological changes in the atherosclerotic intima include increased modification of lipoproteins (e.g., oxidized LDL) and SMC uptake of modified lipoproteins. Foam cell formation by SMCs, in addition to macrophage-derived foam cells, may represent a pivotal

**Figure 1. Accumulation of immature SMC in the arterial intima.** Top panels: SM α-actin is expressed in a wide range of differentiated states of SMC, while mature SMCs produce SM2. In the atherosclerotic rabbit aorta 4 months after mechanical injury and the initiation of a high-cholesterol diet, medial SMC stained positively for SM α-actin and SM2. SM2 was undetectable in intima SMC identified by SM α-actin antibody, indicating their immature phenotype. Bottom panels: Dietary cholesterol lowering for 16 months increased SM2 immunoreactivity in the intima. The data indicate highly plastic nature of SMCs ("re-differentiation"). Alternatively, a repopulation of intimal cells by a more mature SMC subset may have resulted in increased SM2-positive cells, although their origin is unknown.

step in the transition of physiological intimal thickenings into nascent atherosclerotic lesions. [39]. Atherogenic lipoproteins also promote SMC growth by modulating calcium (Ca2+) signaling [40, 41]. SMC foam cell formation may, in part, result from the increased uptake and impaired clearance of lipids [42]. SMCs within intimal thickenings express increased levels of receptors regulating endocytosis of modified lipoproteins, including scavenger receptor A (SR-A), CD36, lectin-type oxidized LDL receptor 1, and low-density lipopro‐ tein receptor-related protein 1 (LRP1) [43]. In parallel, the expression of ATP-binding cassette transporter A1 (ABCA1) and apolipoprotein A1, key molecules for reverse cholesterol transport, decreases in intimal SMCs [39].

**Figure 2. Decreased SMC differentiation in macrophage-rich regions of human coronary arteries.** Immunohisto‐ chemistry of SMC differentiation markers in the left ascending coronary artery (LAD) of a 78-year-old male had devel‐ oped the thickened intima. SM α-actin covers a wide spectrum of SMC differentiation. SMC-specific myosin heavy chains isoforms are definitive markers of differentiated SMC. In particular, SM2 identifies mature SMC. In this athero‐ sclerotic human artery, medial SMC stained positively for all three markers, suggesting an apparently "normal" phe‐ notype. Intima SMC exhibited the diversity of SMC phenotype. Some SM α-actin expressing cells were not immunoreactive for SM1 and SM2 antibodies. Especially, many SMC did not express the detectable level SM2 in the area where many macrophages (CD68) accumulated. They may have undergone phenotypic modulation ("de-differen‐ tiation") due to a pro-inflammatory microenvironment induced by neighboring macrophages. As an alternative possi‐ bility, some of these SM2-negative cells probably co-expressed SM α-actin, SM1, and CD68, an intermediate phenotype between SMC and macrophage lineages. Of note, intraplaque microvessels surrounded by macrophages may have re‐ cruited a subset of activated monocytes as precursor cells for smooth muscle–like cells of the bone marrow origin.

In lipid-laden intimal SMCs, cholesterol accumulation induces cell death. SMC death and subsequent necrosis promote a series of pro-inflammatory events: the release of pro-inflam‐ matory cytokines including monocyte chemo-attractant protein-1 (MCP-1) and IL-1β from the dying and surrounding SMCs [44]; migration and proliferation of adjacent SMCs [45], MCP-1 mediated monocyte infiltration. MCP-1 and IL-1β modulate SMC phenotype, growth, and MMP production [46]. Such a cascade of events accelerates a positive feedback loop of vascular inflammation. To assess the anti-inflammatory and anti-atherosclerotic effects of a monoclonal anti-human IL-1β antibody, a randomized, placebo-controlled trial entitled CANTOS is currently ongoing in high-risk cardiovascular patients [47].