ADDIS ABABA UNIVERSITY

College of Health Sciences, School of Pharmacy

Department of Pharmaceutical Chemistry and Pharmacognosy

A Seminar review on Recent Applications of Hyphenated LC-MS Method in Pharmaceutical Impurity

Analysis; Course Pharmaceutical Literature Survey and Seminar II (Phar 6582)

By: Bizuayehu Belete

Under the Supervision of: Prof. Ariaya Hymete (PhD) and

Mr. Tadele Eticha (MSc, Assoc Prof.) August, 2021

Addis Ababa,1Ethiopia
 Outlines
 Introduction
 Definition and types of impurities
 Methods used for this seminar review
 Hyphenated LC-MS Method in Pharmaceutical Impurity Analysis
 Recent applications of Hyphenated LC-MS Method In Pharmaceutical Impurity Analysis
 Significance of Impurity Profiling And regulatory Requirements For Pharmaceutical Impurities
 Limitations of Hyphenated LC-MS Method In Pharmaceutical Impurities Analysis
 Conclusion

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 INTRODUCTION

 The basic elements of pharmaceutical industry are: Quality, safety and efficacy
 Stability of API & formulation is a 1˚ requirement for pharmaceuticals
 The objective of pharmaceutical industries is:
 Keep the public healthy by enabling patients to get suitable pharmaceutical products

 The safety of a drug is determined by:

 Pharmacological/toxicological profile & adverse effects caused by the impurities
 Formulation should be stable through shelf life
 No drug substance/product is 100% pure, if looks into depth for analysis of impurities

 Impurity of pharmaceuticals may happen at any stage;

 Considering this, guidelines established limits of impurity

(Pounikar et al., 2021; Ayre et al., 2011) 3

 Intro. Cont.…
 Three types of impurities are grouped by (ICH) as;
 Organic, inorganic & residual solvent impurity based on the nature and source of origin

 Toxicity of impurities is the reason behind for:

 Continuous approach to detect and control impurities
 A smattering of impurities does not pose health risks,
 But, some have the potential to damage to human health
 Physiological damage, organ damage and genotoxicity, may be a reason for cancer in humans

 Different pharmacopoeias are set limits to AL of impurities

 Impurities > 0.1% in pharmaceuticals should be reporting, identified and quantified
 In the ICH guidelines, suitable and potent analytical methods are required
(Naveen K., 2021; Singh S., 2022)
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 Intro. Cont.…
 In the near past, higher demand, outstanding improvements & great potential of
applications of modern techniques in pharmaceutical impurities analysis
 In recent years, hyphenated techniques have ever-increasing to solve complex analytical
challenges
 Combining separation tools with spectroscopic techniques for:
 Both qualitative and quantitative analysis of unknown compounds
 Conventional approaches like IR, MS and NMR are successful methods only when there
is less impurity in pharmaceuticals
 Hyphenated LC-MS has significant applications in the analysis of impurities

(Naveen K., 2021; Singh S., 2022; Pilaniya et al., 2010; Wu Y., 2000; Bansi et al., 2005; Kalpesh et al., 2010)
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 Intro. Cont..

 Impurity profiling of an API is a precondition and it is vital importance for:

 Health safety reasons and for regulatory fulfillments

 This seminar focus on:

 Recent application of Hyphenated LC-MS in pharmaceutical impurities analysis

 Significance of hyphenated LC-MS method in impurity analysis and its limitations

(Akshatha et al., 2018, and Vyas et al., 2010)

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 Definition and classification of Impurities

 ICH defines impurities as;

 Any organic material besides the drug substances and excipients

 In pharmaceuticals impurities are unwanted chemicals that remain with the API, or formed during
formulation or upon aging of both API and formulated APIs to medicines

 Impurity profile is:

 Description of known and unknown impurities present in a typical batch of API produced by an accurate
controlled production process

 Impurity profiling includes identification or any qualitative analytical determination

 Impurity profile of a pharmaceutical substance gives maximum possible types of impurities present

 Unwanted chemicals, even in small amount, may affect efficacy & safety of pharmaceutical products
(Ruhela G., and Kaushik D., 2017, and Liu K., and Chen C., 2019)
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 USP has different sections for including impurities in official articles:
 Regular impurities and organic volatile impurities

 USP defined impurity as: foreign substances, toxic impurities, associated components, signal
impurities, ordinary impurities and organic volatile impurities (OVIs) (Bansi et al., 2005)
Classification of Impurities and Sources

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 METHODS

 This seminar review work referred d/t sites & research articles
 It covered limited research works done by different researchers
 Time frame b/n 2019 to now a day (2022)
 D/t research works are included from d/t journals mainly focus on:
 Recent application of hyphenated LC-MS in pharmaceuticals impurities analysis
 Research articles and reviews are googled by using
 ‘Searching with subject headings’ method
 More than 60 articles were googled and downloaded but only 28 articles used

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 Hyphenated LC-MS Method and Pharmaceutical Impurity Analysis

 Outdated analytical methods including HPLC, GC, UV detection, etc., are:

 Become inadequate for effectively handling high challenges in analysis of specific & sensitive products

 Now a day the most common methods used for trace element determination include
 Hyphenated methods (separation technique hyphenated with a detection technique that is more
sensitive)

 Earlier, hyphenated techniques were coupling of separation of a special sample preparation

off-line and later adding a detection technique
 Now a day hyphenated techniques produces:
 Pure fractions of chemical components in a mixture on-line
 Selective information for identification using standards or library spectra

(Joshi et al., 2012) 10

 Hyphenated LC-MS Cont.…

 Hyphenated LC-MS techniques have overcome the challenges and offers;

 Shorter analysis time, higher degree of automation, higher sample through put, better reproducibility,
reduction of contamination because it is a closed system, improved combined selectivity.

 Over the last decades methods improved applications in Pharmaceutical impurities analysis

 Determination of safety and efficacy of API

 It is the most exploited for impurity profiling of drugs/pharmaceuticals

 Has high sensitivity & specificity

(Joshi et al., 2012, and Phatangare et al., 2020)

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 Cont..
 LC-MS is applicable for:
 Identification, quantification and structural characterization of molecules in d/t matrices

 Produces mass spectral data that can give valuable information about:
 Molecular weight, identity, quantity, purity and structure of a pharmaceutical product

 Analyze cpds that lack suitable chromophores not analyzed by other methods

 Assumed as a universal detector for analysis of pharmaceutical product

 Also used to identify components in unresolved chromatographic peaks

 Found in pharmaceutical development activity from research to toxicology studies

 Gives approximately clear structural information about unknown analytes

(Ramachandra B., 2016)

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 1.

Recent Applications of Hyphenated LC-MS In Pharma Impurity Analysis

 Hyphenated LC-MS is:

 An instrument that is more potent than individual parts, LC & MS

 Recent advancements are made in tandem LC-MS such as:

 Triple quadrupole LC-MS (LC-MS/MS) available as a routine technique
 APCI and ESI modes

 Hyphenated LC-MS methods provide:

 Rapid, effective separation of impurities in the form of fragmented ions:
 Help in characterization and structure elucidation of unknown impurities
 Due to high sensitivity, can detect impurities in femtogram level
 Identifying genotoxic impurities in trace levels, ultra-low limit of quantitation, high throughput result
(Ramachandra B., 2016)

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 Cont.…

 Principle of LC-MS involves:

 Separation of the components on an LC column followed by determined the mass-to-charge ratio of an
impurity compound in the MS which gives idea of molecular formulae

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 Cont..

 LC-MS has numerous advantages over individual instrumentation LC and MS

 High selectivity, sensitivity, dynamic range and ruggedness of LC-MS
 It is applicable extensively in different pharmaceutical research areas

 The innovation in the current era

 Increase the application of LC-MS or LC-MS/MS method in impurity analysis
 For rapid investigation of method development for higher sample throughput
 Recent application of hyphenated LC-MS method in pharmaceutical impurities analysis; some research
works are reviewed as follows
(Pounikar et al., 2020)

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 HPLC-MS in Quantitative Determination of Potentially Genotoxic
Impurities in Vildagliptin Drug Material
Vildagliptin (VIL) is an oral hypoglycemic medicine that decreases blood glucose
Several studies showed that;
 4-dimethylaminopyridine, N, N-dimethylaniline, and pyridine are considered highly genotoxic impurities in
the drug material (electrophilic functional groups in their structures)
A novel HPLC-MS method was established to determine impurities in vildagliptin drug product
Sensitivity of the method was excellent at very low levels than the permitted limits
Excellent linearity in the conc. ranges LOQ-150% with coefficients of determination above 0.9990
Recovery in the range of 93.70–108.63%
The method shows good precision, accuracy, sensitivity, selectivity, robustness, and solution
stability
(Al-Sabti B., and Harbali J., 2021) 16
 Quantification of Trace Level 4-Methyl-1-Nitrosopiperazin in Rifampicin Capsules by LC-MS/MS

 Rifampicin is a first-line anti-tuberculosis treatment drug

 US FDA found rifampicin was contaminated with 1-methyl-4-nitrosopiperazine impurity
 FDA had set an acceptable upper limit of 0.16 ppm for MNP impurity in rifampicin
 Though, after considering the benefits and risks of rifampicin for TB patients:
 FDA has temporarily permitted rifampicin impurity at levels < 5 ppm to be circulated

Chemical structures of rifampicin and 1-methyl-4-nitrosopiperazine (MNP) 17

 LC-MS Conditions of the developed method

 Researchers used different parameters and developed method for analysis of MNP impurity
 The method has good linearity, specificity, accuracy, & precision
 LOD & LOQ values for MNP 0.0067 & 0.013 ppm, respectively
 MNP levels in 27 batches of rifampicin samples were determined
 All samples showed MNP levels within the revised FDA-approved upper limit of 5 ppm
(Tao et al., 2022)

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 Impurity Profiling of Ertapenem Analysis by HPLC-MS Method

Ertapenem (ERT); is a carbapenem group of antibiotic, antibacterial towards Gram + and Gram (-)
A compatible MS reverse-phase HPLC approach was successful indicating presence impurities &
degradation product
Eight dimer impurities (m/z 951) and two dehydrated dimers (m/z 933) have been baseline resolved
Chromatographic separation on reverse phase 18 (150 × 4.6 mm, 3.5 μm) using gradient mode
Mobile phase-A was composed of ammonium formate buffer (pH 8), water & acetonitrile, 40:60:01
Mobile phase-B was composed of ammonium formate buffer (pH 8), acetonitrile & methanol, 40:50:10
Flow rate was 1.0 mL min−1 and column was maintained at 40 °C
This method showed comparable selectivity with improved sensitivity for the analysis of those
impurities
(Jadhav et al., 2020)
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 Analyzing Potential Genotoxic Impurities in Pantoprazole Starting
Materials by LC-MS/MS method
 Pantoprazole has approval from the FDA for the treatment of erosive esophagitis
 Developed sensitive and reliable LC-MS/MS method for the quantitative analysis of its impurities
 Starting material of Pantoprazole sodium; 5-difluoro-methoxy-2-mercapto-1H-benzimidazole (DMBZ)

 Analysis was achieved using, a triple quadrupole MS with a positive EI source

 The method was specific, precise, accurate, and linear with in the examined concentration limit
 LOD of the PGIs was < 10 ng/mL and given separation of every impurity from DMBZ
 Quantitative analysis of impurities in DMBZ indicated high performance of the method at a low level
 As a multipurpose and a method is expected to be suitability for DMBZ production and analysis of PGIs
 Method is expected for applicable in-process checking of impurities

(Cheer et al., 2003, and Chen et al., 2020)

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 Screening and Quantification of Nitrosamines Impurity in Sartans
by Multi-Analyte LC-MS/MS Method
 Angiotensin II is a biologically active component that regulate:
 Blood pressure, secretion of aldosterone, and homeostasis of fluid in human body
 An incident of sartan drug contamination was noted in Europe in June 2018
 The drug contaminant was identified as a possible carcinogenic nitrosamine
 To screening and quantification nitrosamine impurities broadly
 A multi-analyte method is required and successfully established and developed
 A feasible and sensitive multi-analyte LC-MS/MS method established
 The method demonstrated a good performance and specificity
 LOD & LOQ for 12 nitrosamines were 20ng/g and 50 ng/g, respectively
 From 557 authentic samples 98 positive samples detected by developed method
(Chang et al., 2020)

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LC-MS/MS Method for Determination and Quantification of Potential Genotoxic Impurities in the Ceritinib API

 Ceritinib is orally administered, most selective and effective anaplastic lymphoma kinase inhibitor
 Few chromatographic methods for determination of impurities in the ceritinib API have been reported
 By LC-MS/MS method quantified 4 PGIs in the ceritinib API

 Chromatographic separation was carried out using:

 C18 column, 0.1% formic acid in water as mobile phase A, and acetonitrile as mobile phase B

 Gradient elution mode at a 0.5 mL min-1flow rate and 3Q mass detection with EI

 The method has good linearity over the conc. range of 0.5–5.0 ppm
 The correlation coefficient obtained > 0.998 and recoveries 83.7 -107.3% for all selected impurities
 The method was capable for quantification of all 4 PGIs
(Antolčić et al., 2020)

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 Simultaneous Determination of Sulfonate Ester Impurities in
PhentolamineMesylate, Amlodipine Besylate, and TosufloxacinTosylate by LC-
APCI-MS/MS
 Sulfonate esters had been identified as potential genotoxic impurities (PGIs) in pharmaceuticals
 Developed and validated LC-MS/MS technique for simultaneous quantification of 15 sulfonate esters
 The method used APCI in MRM mode for quantitation of impurities
 An ODS column as the stationary phase and water-acetonitrile as solvents for gradient elution
 The method has good specificity, linearity, accuracy, precision, and robustness
 Recoveries of the method for sulfonic esters impurities determination 91.6-109.0%
 LOQ in the range of 90.4%-105.2% and LOD < 15ng/mL, 2ng/mL, and 1 ng/mL for methane-sulfonate,
benzenesulfonate, and p-toluene-sulfonate esters impurities, respectively
 The method is sufficiently sensitive to detect the PGIs in these drug tablets
(Jin et al., 2019)

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 Rapid Analysis of Potential Genotoxic Impurities in Rabeprazole Formulations by a
Simple, Sensitive and Straightforward LC-MS Approach

 Rabeprazole is used in the treatment of acid-peptic-associated disease and Helicobacter pylori

 Now a day a sensitive & fully validated LC-MS method is developed
 Separation of impurities are achieved by Symmetry C18 column (100 x 4.6 mm, 3.5 μm)
 Using 0.1% formic acid in water as mobile phase A, and acetonitrile as mobile phase B
 Gradient elution mode at 0.5 mL/min flow rate
 Triple quadrupole mass detector with EI

 The calibration curves of this method shows accurate linearity over the concentration ranges
 The correlation coefficient of the method was > 0.998 and recoveries 94.22%-106.84%
 Furthermore, the proposed method became effective for those impurities analysis
(Yenugu et al., 2018)
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 Other Researches
 In the other research work EI LC-MS with supersonic molecular beams for detection of
untargeted impurities and contaminants analysis in APIs was developed
 EI-LC-MS-SMB used to analyze numerous drug samples spiked with an impurity
 When a molecule is recognized with the help of an EI library like NIST
 The instrument provides EI mass spectra with enhanced molecular ions, known as Cold EI
 Boosts the identification probability

 Ibuprofen and its impurities Analyzed using this method

 NIST library identified both the API and the impurity with names and structures
 The researchers differentiated the method from other methods developed previously
 An EI mode of ionization is an in-vacuum ionization
 While air pressure ionization techniques (ESI, APPI, and APCI) are hindered by matrix effects
(Tsizin et al., 2020)
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 Significance of Impurity Profiling for Pharmaceutical Impurities
 There are different significance of impurity profiling such as;
 Identification and quantification of compounds and helps in control system
 Ensures impurities are within the limits as specific under ICH guidelines
 Improvement of current analytical methods used (origin of impurities)
 Important for development of an alternative route for the synthesis of an API
 Excipients which are not incorporated in the formulation of the API can be monitored
 Potential degradation products can be determined
 To determine stability of polymorphic form of the API (to determine enantiomer impurities)
 Enantiomer impurity profiling confirmed the presence of the correct enantiomer
 Source of genotoxic impurity can be identified (starting material/reagents/catalyst)

(Ruhela G., and Kaushik D., 2017, and Tsizin et al., 2020)

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 Regulatory authorities guidelines
Two general Regulatory authorities guidelines

a. International Council on Harmonization

 Technical requirements for registration of pharmaceutical products for human use
 Regulatory authorities of Europe, Japan and the USA and professionals speak scientific and technical aspects of pharmaceutical
product registration
 The guidelines of impurity profiling in ICH includes;
 impurities in new drug materials Q3A (R2), impurities in new drug merchandise Q3B (R2)
 impurities guideline principle for residual solvents Q3C (R5) and guideline principle for elemental impurities Q3D

b.United States Food and Drug Administration (USFDA)

 A company of USA branch of health and human services, responsible for controlling and supervising safety

 The guidelines of impurity profiling in USFDA includes;

 Impurities in new drug material Q3A, impurities in new drug merchandise Q3B (R2)

 Abbreviated new drug applications impurities in drug substances and ANDAs impurities in drug products

(Ruhela G., and Kaushik D., 2017) 27

 Limitations of Hyphenated LC-MS Method in Pharmaceutical Impurities Analysis

 Works with soft ionization method gives molecular mass information only
 Excipient interference is detected repeatedly
 Solution form drug product generally causes contamination of tip of the capillary
 Confirmation of final structure of identified impurity need NMR spectrometry study
 Factors that should be considered for optimization of the hyphenated LC-MS method
 As mobile phase composition, flow rate, additives like a buffer or any ions pair

 Using ionization techniques should be considered

 Otherwise LC-MS/MS method may be affected

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 Conclusion
 Toxicity of impurities is the reason behind to detect and control in pharmaceuticals

 Interestingly, low amount of impurities does not pose health risks

 While some have the potential to cause significant damage to human health

 Acceptable levels of impurities is limited by d/t pharmacopoeias

 ICH guidelines categories impurities as: organic, inorganic and residual solvents

 On this seminar review finding:

 LC–MS gives information about molecular wgt and structural characterization of impurity

 And also it is applicable for quantification of potential genotoxic impurities in drug substance

 LC-MS/MS method has high Significance of impurity profiling

 In other ways LC-MS/MS method has limitation on the analysis of impurities

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 Thank You
for Your Attention!
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