Impurity Profiling With Use of Hyphenated Techniques

 

Dwivedi Abha*, Kaushik Avinash, Sharma Ganesh N., Akhtar Javed

School of Pharmaceutical Sciences, Jaipur National University, Jagatpura, Jaipur-302017.

*Corresponding Author E-mail: dwivedi1409@hotmail.com

 

ABSTRACT:

Impurity profiling is very important for the identification source, types, structure elucidation and quantitative determination of organic and inorganic impurities in the dosage form and bulk drugs. Presence of these impurities affects the pharmacological property, stability of the formulation. So with the increase demand of the purity of product number of analytical methods have been developed. Identification of the impurities are done by chromatographic, spectroscopic and with the hyphenated techniques. Hyphenated techniques are widely used in impurity profiling as it has revolutionized the impurity profiling by not only the separation but structural identification of the impurities. The hyphenated techniques which are widely used are LC-NMR, LC-MS-MS, GC-MS, and LC-FTIR etc.

 

KEYWORDS Impurity, Impurity Profiling, Hyphenated, Analytical Methods

 


 

INTRODUCTION:

According to the International Conference on Harmonisation (ICH) Guidelines an impurity in a drug substance is any component of the drug substance that is not the chemical entity defined as the drug substance and affects the purity of active ingredient or drug substances. Similarly, an impurity in a drug product is any component of the drug product that is not the chemical entity defined as the drug substance or an excipient in the drug product.1

 

Common terms for impurities:

Intermediate, Penultimate Intermediate and Byproducts : The compounds produced during synthesis of the desired material are called intermediates, especially if they have been isolated and characterized. The penultimate intermediates are the last compound in the synthesis chain prior to the production of the final desired compound. Byproducts are unplanned compounds produced in between the reaction. It may or may not be possible to theorize all of them.2

 

Transformation products: They are very similar to by-products which relates to theorized and non-theorized products that may be produced in the reaction of inactivation or metabolism.3

 

Interaction products: That could occur between various involved chemicals intentionally or unintentionally are generally known as interaction products.4

 

Related products: Products have similar chemical structure and potentially similar biological activity, are generally consider under this category.

Degradation products: These compounds are products due to decomposition of the active ingredient or the material of interest.5

 

Impurity Profiling:

According to the definition of ICH (International Conference on Harmonisation) impurity profile of a drug material is "A description of the identified and unidentified impurities, present in a new drug substance". Impurity profiling is considered to be the common name of analytical activities, the aim of which is the detection, identification/structure elucidation and quantitative determination of organic and inorganic impurities as well as residual solvents in bulk drugs and pharmaceutical formulations.6

 

Classification:

Impurities in drug substance can be classified into the categories like Organic Impurities, Inorganic Impurities, Residual Solvents, and Formulation Related Impurities.

 

Organic Impurities:

This type of impurities in bulk pharmaceutical chemicals those are innocuous by virtue of having no significant undesirable biological activity in the amounts present. Organic impurities can arise during the manufacturing process and/or storage of the drug substance. They can be identified or unidentified, volatile or nonvolatile, and include:

 

Starting materials or intermediates: These are the most common impurities found in every API unless a proper care is taken in every step involved throughout the multi-step synthesis. Although the end products are always washed with solvents, there are always chances of having the residual unreacted starting materials may remain unless the manufacturers are very careful about the impurities.

 

Degradation products: Impurities can also be formed by degradation of the end product during manufacturing of bulk drugs. However, degradation products resulting from storage or formulation to different dosage forms or aging are common impurities in the medicines.

 

Reagents, ligands, and catalysts: These chemicals are less commonly found in APIs; however, in some cases they may poses a problem as impurities.

 

Enantiomeric impurities: The single enantiomeric form of a chiral drug is now considered as an improved chemical entity that may offer a better pharmacological profile and an increased therapeutic index with a more favorable adverse.7

 

Inorganic Impurities:

Inorganic impurities may also derive from the manufacturing processes used for bulk drugs. They are normally known and identified and include the following Reagents, ligands and catalysts.  The chances of having these impurities are rare however, in some processes; these could create a problem unless the manufacturers take proper care during production.

 

Heavy metals: The main sources of heavy metals are the reactors (if stainless steel reactors are used), where acidification or acid hydrolysis takes place and water used in the processes. These impurities of heavy metals can easily be avoided using demineralized water and glass-lined reactors.

 

Other materials: The filters or filtering aids such as centrifuge bags are routinely used in the bulk drugs manufacturing plants, and in many cases, activated carbon is also used. The regular monitoring of fibers and black particles in the bulk drugs is essential to avoid these contaminations. For e.g. filter aids, charcoal.8

 

Residual Solvents:

Residual solvents are organic volatile chemicals used during the manufacturing process or generated during the production. Some solvents that are known to cause toxicity should be avoided in the production of bulk drugs. Depending on the possible risk to human health, residual solvents are divided into three classes.

Class I, viz benzene (2 ppm limit), carbon tetrachloride (4 ppm limit), methylene chloride (600 ppm), methanol (3000 ppm), pyridine (200 ppm), toluene (890 ppm) should be avoided.

 

Class II, viz N, Ndimethylformamide (880 ppm), acetonitrile (410 ppm).

 

Class III, viz acetic acid, ethanol, acetone have permitted daily exposure of 50 mg or less per day, as per the ICH guidelines.

 

A selective gas chromatography (GC) method has been developed to determine the purity of acetone, dichloromethane, methanol and toluene. Using this method, the main contaminants of each organic solvent can be quantified. Moreover, the developed method allows the simultaneous determination of ethanol, isopropanol, chloroform, benzene, acetone, dichloromethane, methanol and toluene with propionitrile as the internal standard.9

 

Crystallization: Formation of the crystals during the crystallization determines the quality and stability of the synthesized molecule. Trace of solvents can be entrapped during the formation of larger size crystals, which may cause the degradation of the drug. Hence, the manufacturers of bulk drugs should take care to produce finer crystals while isolating the products.10

 

Formulation related impurities (Impurities in drug products):

Number of impurities in a drug product can arise out of inert ingredients used to formulate a drug substance. In the process of formulation, a drug substance is subjected to a variety of conditions that can lead to its degradation or other deleterious reaction. Solutions and suspensions are potentially prone to degradation due to hydrolysis. The water used in the formulation cannot only contribute its own impurities; it can also provide a ripe situation for hydrolysis and catalysis. Similar reactions are possible in other solvents that may be used. The formulation related impurities can be classified as follows:

 

Method related A known impurity, 1-(2, 6-diclorophenyl)indolin-2-one is formed in the production of a parenteral dosage form of diclofenac sodium if it is terminally sterilized by autoclave. It was the condition of the autoclave method (i.e., 123 + 2°C) that enforced the intramolecular cyclic reaction of diclofenac sodium forming the indolinone derivative and sodium hydroxide. The formation of this impurity has been found to depend on the initial pH of the formulation. The concentration of the impurity in the resultant product in the ampoule exceeds the limit of the raw material in the BP.11

 

Environmental related: The primary environmental factors that can reduce stability include the following:

I.         Exposures to adverse temperatures: There are many API’s that are labile to heat or tropical temperatures. For example, vitamins as drug substances are very heat-sensitive and degradation frequently leads to loss of potency in vitamin products, especially in liquid formulations.

 

II.         Light-especially UV light: Several studies have reported that ergometrine as well as methyl ergometrine injection is unstable under tropical conditions such as light and heat and a very low level of active ingredient was found in many field samples. In only 50% of the marketed samples of ergometrine injections tested did the level of active ingredient comply with the BP/USP limit of 90% to 110% of the stated content. The custom-made injection of ergometrine (0.2mg/ml) showed almost complete degradation when kept 42 hours in direct sunlight.

 

III.            Humidity: For hygroscopic products, humidity is considered detrimental to both bulk powder and formulated solid dosage forms. Aspirin and ranitidine are classical examples.10,12,13

 

Dosage form related: Although the pharmaceutical companies perform pre-formulation studies, including a stability study, before marketing the products, sometimes the dosage form factors that influence drug stability force the company to recall the product. Fluocinonide Topical Solution USP, 0.05% (Teva Pharmaceuticals USA, Inc., Sellersville, Pennsylvania) in 60 ml bottles, was recalled in the United States because of degradation/impurities leading to sub-potency.14 In general, liquid dosage forms are very much susceptible to both degradation and microbiological contamination. In this regard, water content, pH of the solution/suspension, compatibility of anions and cations, mutual interactions of ingredients and the primary container are critical factors. Microbiological growth resulting from the growth of bacteria, fungi and yeast in a humid and warm environment may result in oral liquid products that are unusable for human consumption. Microbial contaminations may occur during the shelf life and subsequent consumer-use of a multiple-dose product due to inappropriate use of certain preservatives in the preparations15 or because of the semi-permeable nature of primary containers.

 

Mutual interaction amongst ingredients: Most vitamins are very labile and on ageing they pose a problem of instability in different dosage forms, especially in liquid dosage forms. Degradation of vitamins such as folic acid, pantothenic acid, cyanocobalamin and thiamine do not give toxic impurities; however, potency of active ingredients drops below pharmacopoeial specifications. Because of mutual interaction, the presence of nicotinamide in a formulation containing 4 vitamins (nicotinamide, pyridoxine, riboflavin and thiamine) causes the degradation of thiamine to a sub-standard level within a 1-year shelf-life of vitamin B-complex injections. The marketed samples of vitamin B-complex injections were found to have a pH in the range of 2.8-4.0. The custom-made formulation in a simple distilled water vehicle and in a typical formulated vehicle that included disodium edetate and benzyl alcohol was also investigated and similar mutual interaction causing degradation was observed. 16

 

Functional group- related typical degradationEster hydrolysis: Examples included the following: Aspirin, benzocaine, cefotaxime, cocaine echothiophate, ethyl paraben

 

Hydrolysis: Hydrolysis is a common phenomenon for the ester type of drugs, especially in liquid dosage forms. Examples include benzylpenicillin, barbitol, chloramphenicol, chlordiazepoxide, lincomycin and oxazepam.17

 

Oxidative degradation: Hydrocortisone, methotrexate, adinazolam, hydroxyl group directly bonded to an aromatic ring (e.g., phenol derivatives such as catecholamines and morphine), conjugated dienes (e.g., vitamin A and unsaturated free fatty acids), heterocyclic aromatic rings, nitroso and nitrite derivatives and aldehydes (e.g., flavorings) are all susceptible to oxidative degradation.

 

Photolytic cleavage: Pharmaceutical products are exposed to light while being manufactured as a solid or solution, packaged, held in pharmacy shops or hospitals pending use, or held by the consumer pending use.

 

Decarboxylation: Some dissolved carboxylic acids, such as p-aminosalicylic acid, lose carbon dioxide from the carboxyl group when heated. Decarboxylation also occurred in the case of photoreaction of rufloxacin.8

 

Different analytical techniques for impurity profiling:

There are some techniques which are divided 

1.        Reference standard method

2.        Spectroscopic method

3.        Separation method

4.        Isolation method

5.        Characterization method or hyphenated techniques

 

Reference Standard Method:

This Reference standard method is to provide clarity to the overall life cycle, qualification and governance of reference standards used in development and control of new drugs. Reference standards serve as the basis of evaluation of both process and product performance and are the benchmarks for assessment of drug safety for patient consumption. These standards are needed, not only for the active ingredients in dosage forms but also for impurities, degradation products, starting materials, process intermediates, and excipients.18



 


Figure No. 1 Hyphenated Techniques Overview

 

Figure No. 2 GC-MS A Hyphenated Technique

 

Figure No. 3 LC-IR A Hyphenated Technique

 

 


Spectroscopic Method:

The UV, IR, MS, NMR and Raman spectroscopic methods are routinely being used for characterizing impurities.19

 

Separation Method:

The Capillary electrophoresis (CE), Chiral Separations, Gas Chromatography (GC), Supercritical Fluid Chromatography (SFC), TLC, HPTLC, HPLC are regularly being used for separation of impurities and degradation products.20

 

 

Isolation Method:

Chromatographic and non-chromatographic techniques are used for isolation of impurities prior its characterization. A list of methods that can be used for isolation of impurities is given such as Solid-phase extraction methods, Liquid-liquid extraction methods, Accelerated solvent extraction methods, Supercritical fluid extraction, Column chromatography, Flash chromatography, TLC, GC, HPLC, HPTLC, Capillary electrophoresis (CE), Supercritical fluid chromatography (SFC).19

 

Characterization Method and Hyphenated Techniques:

Characterization methods (hyphenated techniques) have highly sophisticated instrumentation. Hyphenated techniques have received ever-increasing attention as the principal means to solve complex analytical problems. The power of combining separation technologies with spectroscopic techniques has been demonstrated over the years for both quantitative and qualitative analysis of unknown compounds in complex products.20

 

To obtain structural information leading to the identification of the compounds present in a crude sample, liquid chromatography (LC), usually a high-performance liquid chromatography (HPLC), gas chromatography (GC), or capillary electrophoresis (CE) is linked to spectroscopic detection techniques, e.g., Fourier-transform infrared (FTIR), photodiode array (PDA) UV-vis absorbance or fluorescence emission, mass spectroscopy (MS), and nuclear magnetic resonance spectroscopy (NMR), resulting in the introduction of various modern hyphenated techniques, e.g., CE-MS, GC-MS, LC-MS, LC-IR and LC-NMR are the most widely used analytical separation techniques for the qualitative and quantitative determination of compounds in products.

 

GC-MS: GC-MS, which is a hyphenated technique developed from the coupling of GC and MS, was the first of its kind to become useful for research and development purposes. Mass spectra obtained by this hyphenated technique offer more structural information based on the interpretation of fragmentations.

 

The fragment ions with different relative abundances can be compared with library spectra. Compounds that are adequately volatile, small, and stable in high temperature in GC conditions can be easily analyzed by GC-MS. Sometimes, polar compounds, especially those with a number of hydroxyl groups, need to be derivatized for GC-MS analysis. The most common derivatization technique is the conversion of the analyte to its trimethylsilyl derivative. In GC-MS, a sample is injected into the injection port of GC device, vaporized, separated in the GC column, analyzed by MS detector, and recorded. The time elapsed between injection and elution is called ''retention time'' (Rt). The equipment used for GC-MS generally consists of an injection port at one end of a metal column (often packed with a sand-like material to promote maximum separation) and a detector (MS) at the other end of the column.

 

Figure No. 4 LC-NMR A Hyphenated Technique

 

LC-IR : The hyphenated technique developed from the coupling of an LC and the detection method infrared spectrometry (IR) or FTIR is known as LC-IR or HPLC-IR. While HPLC is one of the most powerful separation techniques available today, the IR or FTIR is a useful spectroscopic technique for the identification of organic compounds, because in the mid-IR region the structures of organic compounds have many absorption bands that are characteristic of particular functionalities, e.g., -OH, -COOH, and so on. However, combination of HPLC and IR is difficult and the progress in this hyphenated technique is extremely slow because the hyphenated technique's 237 absorption bands of the mobile phase solvent are so huge in the mid-IR region that they often obscure the small signal generated by the sample components.

 


 

Table no. 1: Hyphenated Techniques with examples of drugs with analysis

Hyphenated Techniques

Example

Author

1.        GC–MS

Methamphetamine (Ephedrine & Pseudoephedrine is major component in it which is due to Aziridine intermediate.)

Lekskulchai et al 21

2.        HPLC–MS & HPLC–NMR

Acarbose

Novak et al 22,23

5-aminosalicylic acid

Novak et al 24

Pseudoephedrine sulphate (2-carboxyamino-propiophenone and 2-formyl-2-methylamino-acetophenone impurities)

Wu et al 25

3.        LC-MS-MS

Emtricitabine (Methyl methanesulfonate and Ethyl methanesulfonate impurities)

Kakadiya et al26

4.        CE-MS

cis-Ketoconazole

Maria et al27

5.        LC-MS

Tandospirone

Meiling et al28

6.        LC-NMR

Fluticasone propionate

Mistry et al29

7.        HPLC-DAD/UV-ESI/MS

Lumefantrine

Verbeken et al30

8.        TLC- Densitometer

Chlorpromazine Hydrochloride

Trifluoperazine Dihydrochloride

Promazine Hydrochloride

Doxipen Hydrochloride

Maslanka et al31

9.        HPLC-DAD-FTIR

Tropicamide

Stefanowicz et al32

10.     HPLC- LC/MS/MS

Piperaquine phosphate

Dongre et al33

11.     HPLC-DAD/LC-ESI-MS

Doxipan (Doxycycline and related impurities)

Fiori et al34

 


LC-MS: LC-MS refers to the coupling of an LC with a mass spectrometer (MS). The separated sample emerging from the column can be identified on the basis of its mass spectral data. A switching valve can help make a working combination of the two techniques. A typical automated LC-MS system consists of double three-way diverter in-line with an autosampler, an LC system, and the mass spectrometer. The diverter generally operates as an automatic switching valve to divert undesired portions of the eluate from the LC system to waste before the sample enters the MS.

 

LC-NMR: Among the spectroscopic techniques available to date, NMR is probably the least sensitive, and yet it provides the most useful structural information toward the structure elucidation of natural products. Technological developments have allowed the direct parallel coupling of HPLC systems to NMR, giving rise to the new practical technique HPLC-NMR or LC-NMR, which has been widely known for more than last 15 years. The first on-line HPLC-NMR experiment using superconducting magnets was reported in the early 1980s. However, the use of this hyphenated technique in the analytical laboratories started in the latter part of the 1990s only. LC-NMR promises to be of great value in the analysis of complex mixtures of all types, particularly the analysis of natural products and drug-related metabolites in biofluids.

 

CE-MS: CE is an automated separation technique introduced in the early 1990s. CE analysis is driven by an electric field, performed in narrow tubes, and can result in the rapid separation of many hundreds of different compounds. The versatility and the many ways that CE can be used mean that almost all molecules can be separated using this powerful method. It separates species by applying voltage across buffer-filled capillaries, and is generally used for separating ions that move at different speeds when voltage is applied, depending on their size and charge. The solutes are seen as peaks as they pass through the detector and the area of each peak is proportional to their concentration, which allows quantitative determinations. Analysis includes purity determination, assays, and trace level determinations. When an MS detector is linked to a CE system for acquiring on-line MS data of the separated compound, the resulting combination is termed as CE-MS.35

 

CONCLUSION:

Impurity profiling of the drug substance is very important during the synthesis and after the manufacturing of the dosage form. As it provide the information related to its type, toxicity, several organic and inorganic impurity that usually affect on the formulation. Certain impurity present in the dosage form doesn’t hinder the therapeutic value but affect the stability and other properties. Impurity profiling can be done by the number of analytical techniques like chromatography (GC, LC, HPLC), Spectroscopic technique (MS, NMR, UV). But now a day’s hyphenated technique is used which are developed by the coupling of separation and spectroscopic technique (LC-MS, HPLC-NMR, LC-FTIR). It provides the adequate result of analysis crude drug product and determination of the impurity in the dosage form.

 

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2.        U.S. Food and Drug Administration. Guidance for Industry, Q3A Impurities in New Drug Products. June 2008.

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11.     Roy J, Islam M, Khan A.H., Das S.C., Akhteruzzaman M, Deb A.K, Alam, A.H.M. Diclofenac Sodium Injection Sterilized By Autoclave And The Occurrence Of Cyclic Reaction Producing A Small Amount Of Impurity. J Pharm Sci. 2001, 90, 541-544.

12.     Walker G.J.A, Hogerzeil H.V, Hillgreen U. Potency of Ergometrine in Tropical Countries. Lancet. 1988; 2: 393.

13.     Hogerzeil H.V, Battersby A, Srdanovic V, Stjernstrom N.E. Stability Of Essential Drugs During Shipment To The Tropics. British Medical Journal 1992; 304: 210-214.

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15.     Hoq M.M, Morsheda S.B, Gomes D.J. Development of Appropriate Preservative System for Liquid Antacid: Bacterial Contaminants in Antacid Samples. J. Microbiology 1991; 8(1): 5-9.

16.     Sapra A, Kakkar S, Narasimhan B. Source of Impurities – A Review. International Research Journal of Pharmacy 2012; 3(1): 57-59.

17.     Jiben Roy. Pharmaceutical Impurities - A Mini Review. AAPS PharmSciTech 2002; 3(2): 1-8.

18.     Bari S.B, Kadam B.R, Jaiswal Y.S, Shirkhedkar A.A. Impurity profile: Significance in Active Pharmaceutical Ingredient. Eurasian Journal of Analytical Chemistry 2007; 2(1): 32-53.

19.     Chatwal GR. Pharmaceutical Inorganic Chemistry. Himalaya, New Delhi; 1991: 1.

20.     Ahuja S. Impurities Evaluation of Pharmaceuticals. Marcel Dekker, New York 1998; 142.

21.     Lekskulchai V, Carter K, Poklis A, Soine W. GC-MS Analysis of Methamphetamine Impurities: Reactivity of (+)-or (–)-Chloroephedrine and cis- or trans-1,2-Dimethyl-3-phenylaziridine. J Anal Toxicol 2000; 24: 602-605.

22.     Novak P, Tepes P, Cindric M, Ilijas M, Dragojevic S, Mihaljevic K. Combined Use Of Liquid Chromatography-Nuclear Magnetic Resonance Spectroscopy And Liquid Chromatography-Mass Spectrometry For The Characterization Of An Acarbose Degradation Product. Journal of Chromatography A, 2004;  1033(2): 299-303.

23.     Novak P, Cindric M, Tepes P, Dragojevic S, Ilijas M, Mihaljevic K. Identification of impurities in acarbose by using an integrated liquid chromatography-nuclear magnetic resonance and liquid chromatography-mass spectrometry approach.Journal of Separation Science 2005; 28(12): 1442-1447.

24.     Novak P, Tepes P, Fistric I, Bratos I, Gabelica V. The Application of LC-NMR and LC-MS for The Sepration and Rapid Structure Elucidation of An Unknown Impurity in 5-aminosalicylic acid J. Pharm. Biomed. Anal. 2006; 40(5): 1268.

25.     Wu N, Feng W, Lin E, Chen G, Patel J, Chan T.M, Pramanik B. Quantitative And Structural Determination Of Pseudoephedrine Sulfate And Its Related Compounds In Pharmaceutical Preparations Using High-Performance Liquid Chromatography. Journal of Pharmaceutical and Biomedical Analysis 2002; 30(4): 1143.

26.     Kakadiya P.R, Chandrashekhar T.G, Ganguly S, Singh. D.K, Singh V. Low Level Determination Of Methyl Methanesulfonate And Ethyl Methanesulfonate Impurity In Emtricitabine Active Pharmaceutical Ingredient By LC-MS-MS Using Electrospray Ionization. Analytical Chemistry Insights 2011; 6: 21-28.

27.     Maria L.M, Maria C.P, Carmen G.R, Alejandro C., Antoniol C. Identification And Quantitation Of Cis-Ketoconazole Impurity By Capillary Zone Electrophoresis–Mass Spectrometry. Journal Of Chromatography 2006; 1114(1): 170-177.

28.     Qi Meiling, Wang Peng, Xiao Goekeng. Determination Of Tandospirone And Its Impurities In Drug Formulations By LC-UV And LC-MS.Chromatographia 2004; 59(5-6): 373-379.

29.     Mistry Nisha, Ismail I.M, Smith M.S, Nicholson J.K, Lindon J.C. Characterization Of Impurities In Bulk Drug Batches Of Fluticasone Propionate Using Directly Coupled HPLC–NMR Spectroscopy And HPLC–MS. Journal Of Pharmaceutical And Biomedical Analysis 1992; 16(4): 697-704.

30.     Verbeken M, Suleman S, Baert B, Vangheluwe E, Dorpe S.V, Burvenich C, Duchateau L, Jansen F.H, Spiegeleer B.D. Stability Indicating HPLC-DAD/UV-ESI/MS Profiling Of The Anti-Malarial Drug Lumefantrine. Malar. J. 2011; 10: 51.

31.     Maslanka Anna, Krzek Jan. Use Of TLC With Densitometer Detection For Determination Of Impurity In Chlorpromazine Hydrochloride, Trifluoperazine Dihydrochloride, Promazine Hydrochloride, Doxipen Hydrochloride. Journal Of Planar Chromatography-Modern TLC 2007; 20(6): 463-475.

32.     Stefanowicz Z, Stefanowicz J, Mulas K. Determination Of Tropicamide And Its Major Impurity In Raw Material By The HPLC-DAD Analysis And Identification Of This Impurity Using The Off-Line HPLC–FT-IR Coupling. Journal Of Pharmaceutical And Biomedical Analysis.2009.49 (2).214-220.

33.     Dongre V, Karmuse P.P, Ghugare P.D, Gupta M, Nerurkar B, Shaha C, Kumar A, Characterization And Quantitative Determination Of Impurities In Piperaquine Phosphate By HPLC And LC/MS/MS. Journal Of Pharmaceutical And Biomedical Analysis 2007; 43(1): 186-195.

34.     Fiori J, Grassigi G, Filippi P, Gotti R, Cavrini V. HPLD-DAD And LC-ESI-MS Analysis Of Doxycycline And Related Impurities In Doxipan Mix, A Medicated Premix For Incorporation In Medicated Feedstuff. Journal Of Pharmaceutical And Biomedical Analysis 2005; 37(5): 979-985.

35.     Patel K.N, Patel J.K, Patel M.P, Rajput G.C, Patel H.A. Introduction To Hyphenated Techniques And Their Applications In Pharmacy. Pharmaceutical Methods 2010: 1(1); 2-13.

 

 

 

Received on 13.05.2012        Modified on 24.05.2012

Accepted on 20.06.2012        © AJRC All right reserved

Asian J. Research Chem. 5(7): July, 2012; Page 875-881