INTRODUCTION:

For the drug development and formulation process detecting and quantifying drug substances and their impurities in raw materials and final product testing is an essential part. Impurities may influence the safety and efficacy of the pharmaceutical products. Impurity profiling is becoming essential factor and required for regulatory compliance for pharmaceutical development program. Stress testing of the drug substance can help in the identification of degradation products which can help in establishing the degradation pathways and to investigate the stability-indicating power of the analytical procedures applied for the drug substance and drug product.

A stability-indicating method is an analytical procedure that is capable of discriminating between the active pharmaceutical ingredients (API) from any degradation product formed under defined storage conditions during the stability evaluation period. In addition, it must also be sufficiently sensitive to detect and quantify one or more degradation products. ^[1-7] The objectives of this review paper are to discuss the role of various hyphenated analytical techniques such as HPLC-UV, HPLC-MS, GC-MS and UPLC-MS/MS for identification of impurities and degradation products in pharmaceutical products.

Systematic Approach for Stress Testing:

A systematic approach is now developed for stress testing of pharmaceutical products which can generate the degradation in most efficient way. The stress conditions employed for the degradation study includes acid hydrolysis (1 N HCl), alkali hydrolysis (1 N NaOH), oxidation (3% H₂O₂), and light (carried out as per ICH Q1B). For acid, alkali hydrolysis, and oxidation, the study period is 1 h whereas for light study period is 24 h. All stress conditions employed for forced degradation studies are initially carried out at room temperature (25°C). It should include the effect of temperatures in 10°C increments (e.g., 50°C, 60°C etc.) above that of accelerated testing, humidity (e.g., 75% RH or greater), acid-base hydrolysis, oxidation, and photolysis. Stress testing should induce not more than 5-15 % degradation of the main compound. Stress conditions employed for forced degradation study as per ICHQ1A (R2) is shown in Figure 1.

Figure 1: Stress conditions employed for forced degradation study as per ICHQ1A (R2)

The most common hyphenated analytical technique for monitoring forced degradation experiments is HPLC with UV and/or MS detection for peak purity, mass balance, and identification of degradation products. Combining the chromatographic speed, resolution, and sensitivity of ultra-performance liquid chromatography (UPLC) separations with the high-speed scan rates of UPLC-specific photodiode array (PDA) and quadruple time-of-flight mass spectrometry (QTOF-MS/MS) detection will provide thorough identification of degradation products and shortening the time required to develop stability-indicating methods.^[8-14] In this stage stress testing is applied to the drug substance as pure form and also its dosage form. The degradation study is done by applying the acid-base hydrolysis, oxidation and photolysis. The generated degradation products are identified by suitable analytical technique and simultaneously degradation pathway is established. The results of stress testing are ultimately helpful in the determination of shelf lives of drug products. Information provided by stress testing at different stages of pharmaceutical development is given in Table 1.

Table 1. Information Provided by Stress Testing at Various Stages

Development Stages

Purpose of stress testing

Pre-formulation

1. Selection of compounds and excipients.

2. Formulation optimization.

3. Selection of proper packaging.

4. Registration application dossiers.

Formulation, registration batches and manufacturing

1. Stability-indicating analytical method.

2. Understanding impurity profile.

3. Structure elucidation of degradation products.

4. Establishment of degradation pathways.

5. Establishment of shelf life.

6. Selection of packaging and storage instructions.

Impurities in Pharmaceutical Products:

Impurities in pharmaceuticals are the unwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during formulation, or upon storage of both API and formulated APIs. ICH guidelines classify impurities into three categories: organic impurities, inorganic impurities, and residual solvents. These impurities can be from a variety of sources, as given in Table 2.

Table 2. Impurity Classification Based on ICH Guidelines

Nature of Impurities	Sources
Organic Impurities	· Starting materials · Intermediates · Related products · Degradation products
Inorganic Impurities	· Heavy metals · Inorganic salts
Residual solvents	· Organic or inorganic liquids

According to the ICH guidelines impurities in new drug product, degradation products observed in stability studies conducted at recommended storage conditions should be identified when present at a level greater than the identification thresholds (0.1% for a maximum daily dose of >2g). Identification of impurities below the 0.1% level is generally not considered to be necessary unless the potential impurities are expected to be unusually potent or toxic. For the drug development and formulation process detecting and quantifying drug substances and their impurities in raw materials and final product testing is an essential part. Impurities may influence the safety and efficacy of the pharmaceutical products. An easy way of doing this is to compare the retention times of known process-related compounds to that in question. If this analysis confirms that the compound is an unknown, the next step would be to obtain an LC-MS on the compound. Mass spectrometry provides structural information which aids in determining structure. In some cases, mass spectrometry will be enough to identify the compound. In other cases, more complicated methods like liquid chromatography coupled to nuclear magnetic resonance (LC-NMR) are needed or the impurity will need to be isolated in order to obtain additional information. ^[15-20]

Identification of Impurities/Degradation Products by HPLC-UV:

Once a decision has been made to identify an unknown compound, the next step is to evaluate all known process-related impurities, precursors, intermediates, and degradation products. By observing the relative retention times (HPLC) of all known process-related impurities, precursors, and intermediates, one can quickly determine whether or not the impurity of interest is truly unknown. If the relative retention time of the unknown impurity matches that of a standard, then it can be identified using HPLC with UV or photodiode array detection and LC-MS, and GC-MS for volatile impurities. The identity is confirmed by correlating the retention time, ultraviolet spectra, and mass spectra of the unknown impurity with that standard. The process outlined in Figure 2. Illustrates the overall strategy used for identification of unknown impurities and degradation products. If the relative retention time does not match that of a standard. The next step is to obtain molecular mass and fragmentation data via HPLC-MS. It is essential to determine the molecular mass of the unknown impurity. To run LC-MS, a mass spectrometry-compatible HPLC method must be developed. If the mass spectrometry data evaluation yields sufficient structural information, this eliminates the need to isolate the impurity. If standards are not available, which is usually the case, the proposed structures can be discussed with the project team. The project team can then decide if the information is suitable for their needs, or if isolation is required. A number of methods can be used for isolating impurities and/or degradation products. Three of the most utilized techniques are TLC, flash chromatography (column chromatography), and preparative HPLC. ^[21-23]

Identification of Impurities/Degradation Products by HPLC-MS:

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. In the technique of mass spectrometry, the compound under investigation is bombarded with a beam of electrons which produce an ionic molecule or ionic fragments of the original species. The resulting assortment of charged particles is then separated according to their masses. The spectrum produced, known as mass spectrum is a record of information regarding various masses produced and their relative abundances. Mass spectrometry is one of the best methods to detect impurities. Impurities present can be detected by the additional peaks, highest value of mass peaks than compound itself and from the fragmentation pattern. Mass spectrometry by itself and in various combinations with other analytical instrumentation, is the first logical technique to use to identify and detect unknown structures. Mass spectrometry requires small amounts of sample to obtain significant amounts of structural information on the target compound. One of the powerful tools of impurity profile is liquid chromatography (LC) coupled with mass spectroscopy (MS), and it is employed for the identification of impurities, natural products, drug metabolites, and proteins. LC-MS is steadily applied to scrutinize impurity during pharmaceutical product development and manufacturing process to support the safety evaluation of batches used in clinical studies. After a step-by-step investigation using various LC-MS techniques, it is often possible to propose a possible structure for an unknown impurity. The quadrupole mass analyzer is very popular for LC-MS, due to its relative simplicity and relatively low cost. Systematic line diagram for isolation and identification of impurities is shown in Figure 2. The primary information available from a LC-MS experiment using a single quadrupole analyzer is the molecular weight of the unknown impurity. Molecular weight is the most important structural information required for unknown structure elucidation. ^[24-26]

Identification of Impurities by GC-MS:

Gas chromatography coupled with mass spectrometry is particularly useful in the determination of organic volatile impurities. In this technique the carrier gases such as hydrogen, helium, nitrogen and argon used as mobile phase for chromatographic separation. The samples are converted in to vapour form by heating at higher temperature, mixed with carrier gas and detected by mass spectrometry. ^[27]

Identification of Impurities/Degradation Products by UPLC/Q-TOF-MS:

Ultra-performance liquid chromatography (UPLC) has been investigated as an alternative to HPLC for the analysis of pharmaceutical development compounds and particularly very sensitive technique for identification of degradation products and drug metabolites. UPLC produced significant improvements in method sensitivity, speed, and resolution. Acquity UPLC is specially designed to resist higher back-pressures, with the advantages of fast injection cycles, low injection volumes, and temperature control (4-40ºC), which collectively contributes to speedy and sensitive analysis. Furthermore, Acquity UPLC columns contain hybrid X-Terra sorbent, which utilizes bridged ethylsiloxane/silica hybrid (BEH) structure, ensures the column stability under the high pressure and wide pH range (1-12). UPLC is a novel chromatographic technique utilizing high linear velocities, which is based on concept using columns with smaller packing (1.7-1.8 μm porous particles) and operated under high pressure (up to 15000 psi). This is an extremely powerful approach which dramatically improves peak resolution, sensitivity and speed of analysis. In addition to UPLC, the use of orthogonal quadrupole time-of-flight mass spectrometry (Q-TOF-MS) with low and high collision energy full scans acquisition simultaneously performed, allows the generation of mass information with higher accuracy and precision, which is ultimately helpful in structure elucidation and identification of fragmentation pattern of the compounds. It also confidently detects impurities in compounds even at trace levels. The UPLC/Q-TOF-MS has been successfully applied in the simultaneous determination of aceclofenac and paracetamol and their degradation products in tablets. In the reported study, the fragementation pattern and forced degradation studies on aceclofenac and paracetamol were carried out. ^[14] The sensitivity and flexibility of exact mass time-of-flight mass spectrometry with alternating collision cell energies, combined with the high resolving power of the UPLC system, allows for the rapid profiling and identification of impurities and/or degradation products. Its most popular applications are in drug discovery, samples characterization, structural elucidation, etc., determination of degradation products, impurities, by-products, break-down products, stability testing, etc., where accurate mass determinations are required. Also it is used more widely in in-vitro and in-vivo bioanalytical samples for metabolite research and identification with accurate mass. Time-of-flight (TOF) is one of the most widely used mass analyzers for accurate mass measurement using LC-MS. In a TOF analyzer, the mass of an ion is determined based on the time it takes to reach a detector through an evacuated flight tube. Because of this feature, TOF is an ideal mass analyzer for large biomolecules that are ionized by MALDI (Matrix assisted laser desorption ionization. ^[28-30]

CONCLUSION:

Hyphenated chromatographic techniques are becoming most popular in the identification of impurities and degradation products in pharmaceutical products. Each and every hyphenated technique has its own specific application. Selection of technique should be done in such a way that it should provide simple, fast, sensitive, specific, accurate and economic analytical method. From the above discussion, it can be concluded that the systematic approach and strategy is required for while employing these techniques for identification and evaluation of impurities and degradation products.

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Received on 03.01.2017 Accepted on 02.02.2017

Asian J. Pharm. Ana. 2017; 7(1): 31-35.

DOI: 10.5958/2231-5675.2017.00006.0