a review on: separation of different process related...

JPR:BioMedRx: An International Journal Vol.1 Issue 7.June 2013

Desai Sagar S et al. /JPR:BioMedRx: An International Journal 2013,1(7),673-684

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Review Article Available online throughhttp://jprsolutions.info

*Corresponding author.Desai Sagar SDepartment of Pharmaceutical Chemistry,Amrutvahini college of Pharmacy,Sangamner, Dist: Ahmednagar,Maharastra, India

A Review On: Separation of different process relatedimpurity in tablet formulation of drug

Desai Sagar S*, Dr. V. K. Deshmukh, Dr. S. R ChaudhariDepartment of Pharmaceutical Chemistry, Amrutvahini college of Pharmacy, Sangamner, Dist: Ahmednagar,Maharastra, India

Received on:14-03-2013; Revised on:22-04-2013; Accepted on:27-05-2013

ABSTRACTAs new molecular entities are evaluated in clinical development the understanding, identification, quantification and control of impurities indrug substances are essential. As chemical processes used to produce drug substances mature from the early phases of developmentthrough registration, a concomitant maturing of process-related impurity understanding and control is required. Tablet formulations whichare used in different diseases condition containing impurity as per ICH guidelines limit. This paper outlines the separation of different processrelated impurity in tablet formulation. All authors develop the analytical method for separation and increase resolution of different processrelated impurity with active pharmaceutical ingredient in tablet formulation.

Key word: Related substances, residual solvents, metallic catalysts, toxic impurities.

ISSN: 2321-4988

INTRODUCTION

Impurity profilingImpurity profile of Active Pharmaceutical Ingredient (API) gives anaccount of impurities present in it. Impurity profile is a description ofthe identified and unidentified impurities present in a typical batchof API produced by a specific controlled production process. Regu-latory authorities such as USFDA (United States Food and DrugAdministration), cGMP (Current Good Manufacturing Practice) andMCA (Ministry of corporate Affairs) insist on the impurity profilingof drugs.

There is an ever increasing interest in impurities present in API’s.Recently, not only purity profile but also impurity profile has becomeessential as per regulatory requirements. In the pharmaceutical world,an impurity is considered as any other organic material, besides thedrug substance, or ingredients, arising out of synthesis or unwantedchemicals that remain with API’s. The impurity may be developedeither during formulation, or upon aging of both API’s and formu-lated API’s in medicines [1]. Impurity profile of a substance underinvestigation gives maximum possible types of impurities presentin it. It also estimates the actual amount of different kinds of impuri-ties present in it. The impurity profile is normally dependent upon theprocess or origin of the API.

Identification, qualification and control of impurities in the drugsubstance and drug product, are an important part of drugdevelopment and regulatory assessment. Qualification of theimpurities is the process of acquiring and evaluating data thatestablishes biological safety of an individual impurity or a given im-purity profile at the level specified. Extraction, column chromatogra-phy and preparative separations, etc. are methods generally used forisolation of impurities.

The spectroscopic studies conducted to characterize the structureof actual impurities present in the drug substance above anapparent level of 0.1% should be described. Hyphenated methodssuch as gas chromatography, mass spectroscopy, or liquidChromatography and the number of other chromatographic methodsare perfectly suitable for the characterization of impurities. During thecourse of drug development studies the qualitative degradation pro-file of a new drug product may change, resulting in new degradationproducts that exceed the identification or qualification threshold. Inthis event, these new degradation products should be identified orqualified [2].

In I.C.H. Guidelines impurities in new drug substances are addressedfrom two aspects:1. Chemistry aspects include classification and identification ofimpurities, report generation, setting specification and a brief dis-cussion of analytical procedures.2. Safety aspects include specific guidance for qualifying impuritiesthat were not present in batches of new drug substance used insafetyand clinical studies.



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2. ICH terminology:According to ICH guidelines, impurities in the drug substanceproduced by chemical synthesis can broadly classified into:

1. Organic impurities, (process and drug related)2. Inorganic impurities,3. Residual solvents.

Organic impurities may arise during the manufacturing process and

Impurity [2]

1. Impurity is defined as any substance coexisting with the origi-nal drug, such as starting material or intermediates or that is formed,due to any side reactions.2. In the pharmaceutical world, an impurity is considered as anyother organic material, besides the drug substance, or ingredients,arising out of synthesis or unwanted chemicals that remain withAPI’s.3. Impurities can also be formed by degradation of the endproduct during manufacturing of the bulk drugs.

Types of impurity [3]

Impurity can be of three types:1. Impurities closely related to the product and coming from thechemical or from the biosynthetic route itself,2. Impurities formed due to spontaneous decomposition of the drugduring the storage or on exposure to extreme conditions,3. The precursor that may be present in the final product as impuri-ties.

Common terms for impurities [3]

1. Intermediate, penultimate intermediate and by products,2. Transformation products,3. Interaction product,4. Related product,5. Degradation product.

Sources of impurities in drug productsVarious types of impurities that may be present in pharmaceuticalsubstance can come from the following sources:

1. Raw materials used,2. Method of manufacture adopted,3. Due to the instability of product and4. From the atmospheric contaminants.

Classification of impurities [4]

1. According to United States Pharmacopoeia (USP):

Impurities1. Impurities in Official Articles2. Ordinary Impurity3. Organic volatile impurities

or storage of the drug substance may be identified or unidentified,volatile or non-volatile, and may include:1. Starting materials or intermediates,2. By-products,3. Degradation products,4. Intermediates,5. Reagents, ligands, and catalysts.

Inorganic impurities can result from the manufacturing process. Theyare normally known and identified and include:

1. Heavy metals or other residual metals,2. Inorganic salts,3. Other material (e.g., filter aids, charcoal)

ICH limits for impurities [4]

According to ICH guidelines on impurities in new drug products,identification of impurities below 0.1% level is not considered to benecessary, unless potential impurities are expected to be unusuallypotent or toxic. According to ICH, the maximum daily dose qualifica-tion threshold to be considered is as follows; < 2g/day 0.1 % or 1 mgper day intake (whichever is lower) >2g/day 0.05%.

In summary, the new drug substance specifications should include,limits for:1. Organic Impurities- Each specific identified impurity- Each specific unidentified impurity at or above 0.1%- Any unspecific impurity, with limit of not more than 0.1%

- Total impurities2. Residual solvents3. Inorganic impurities

Impurity type Impurity source

1.Process-related drug substance OrganicStarting materialIntermediateBy-productsImpurity in starting material

2. Process-related drug products Organic or inorganicReagents, catalysts, etc.

3. Degradation drug substance or drug products OrganicDegradation products

4. Degradation drug products Excipient interaction

Tablet Pharmaceutical dosage form

Table 1. Description of impurity types and their sources

Antihypertensive

R. Nageswara Rao et al., Development of a validated liquid chro-matographic method for determination of related substances oftelmisartan in bulk drugs and formulations [5]. The EP method usessodium pentanesulfonate monohydrate an ion pairing reagent as amobile phase additive phase which decreases the column life andneeds a long time for equilibration.



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TLM and its related substances are polar as well as basic in nature. Itcould be due to the strong interaction between amino groups and thesurface hydroxyls on the silica used as stationary phase which makesdifficult to elute the compounds from the column. Therefore a re-verse-phase chromatographic system was selected. The chromato-

NH2

H3CO

CH3

Cl CH3

O

+

V

NH

H3CO

O

CH3

CH3

ONO2

HNO3, H2SO4H2, PdC

NH

H3CO

O

CH3

CH3

ONH2

Methanol

AcOH

N

N CH3H3CO

O H

CH3

AcOH

N

NH

CH3OH

O

CH3

N

NH

CH3

N

N

CH3

CH3NH2

NH

CH3

Phosphoric acid/150~1550C/4

-5h

II

N

C H

C H 3

N

N

C H 3

C H 3

H 3CO

O

KOH flakes/CH 3 CN/80-850C/1.5

-2 h.

N

C H

C H 3

N

N

C H 3

C H 3

OH

O

graphic separation was achieved on Lichrospher RP-18 column (250× 4.6 mm, 5 _m), using 20 mM ammonium acetate containing 0.1% (v/v) triethylamine (pH adjusted to 3.0 with trifluoroacetic acid) andacetonitrile as mobile phase at 25

0C. The detection was performed at

254 nm. All the impurities (I-VII) are process related impurities.

VII TelmisartanFigure 1. Synthesis of telmisartan and process related impurity (I–VII in order of their chromatographic elution)

NH

H3CO

O

CH3

CH3

O C6H5Cl

VI IV

III II

OCH 3

O

Br

KOH flakes/acetone/25~350C/1.5

-2 h.

Methanolic HCl (30%)/methanol/acetonitrile



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Figure 2. A typical HPLC chromatogram of TLM spiked with 10%of related substances I–VII, obtained under optimized conditions.

Svetlana Milovanovic et al., Development and validation of reversedphase high performance liquid chromatographic method for determi-nation of moxonidine in the presence of its impurities [6]. Used sym-metry shield C18 column (250 mm× 4.6 mm, 5 µm) for the analysis ofmoxonidine and its impurities in tablet formulations by employing amobile phase consisting of methanol–potassium phosphate buffer(0.05 M) mixture (15:85, v/v) (pH 3.5) at a flow rate of 1 ml min-1;detection at 255 nm. The column temperature was set at 250C.Moxogamma 0.4® tablet is used to determine monoxidine in the pres-ence of impurity.

During the method development, the critical separation pairs wereidentified and they were formed with Impurities C and D as well aswith Impurity A and moxonidine. Polar hydroxyl groups on a C-4position of pyrimidine rings in the structures of Impurities C and Dprovided earlier elution comparing with other components. At thesame time, chlorine atom on a C-4 position of pyrimidine rings in thestructures of moxonidine and Impurity A caused prolonged retention.Since moxonidine and Impurity C contain lipophilic methoxy groupsat C-6 position in the pyrimidine ring, they remained slightly longeron stationary phase comparing to other component in the critical pair.Therefore, it was quite reasonable that Impurity B, having methoxygroups on both C-4 and C-6 positions of the pyrimidine ring, hadbeen the last eluted component. The main advantages of this methodcompared with the official Ph. Eur. method are avoiding ion-pair re-agent and column heating, as well as significantly shortening of runtime.

N

N

CH3

NH

NNHCH3

Cl N

N

CH3

NH

NNHOCH3

OCH3N

N

CH3

NH

NNHCl

Cl

(A) (B) (C)

N

N

CH3

NH

NNHCl

OHN

N

CH3

NH

NNHOCH 3

OH

(D) (E)Figure 3. Structure of moxonidine (A) and Impurity A (B), B (C), C (D)and D (E).

Figure 4. The representative chromatogram of moxonidine with im-purities

Figure 5. Chromatogram of Nebivolol and its impurity

Ediga Sasi Kiran Goud et al., RP-HPLC validation of related sub-stances of nebivolol in bulk & 2.5/5/10/20 mg tablets [7]. A gradientreverse phase HPLC method was developed for the determination ofrelated substances in Nebivolol in bulk and their tablets. The methodwas carried out on a Kinetex C18 column (75 x 4.6 mm; 2.6µ) using amobile phase mixture of buffer pH 3.4 (tetra butyl ammonium hydro-gen sulphate), acetonitrile and water (95:5) in a gradient elution at aflow rate of 1.0ml/min at wavelength of 280 nm. The impurities sepa-rated with a RRT of 0.9 for desfluoro, 1.06 for related impurity-A and1.27 for benzylated impurity with respect to Nebivolol. Sample prepa-ration containing standard preparation for 2.5, 5, 10, 20mg of nebivololwith placebo preparation. The method fulfilled the validation criteria:specificity, linearity, accuracy, precision, limit of detection and limit ofquantitation.



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Khalid. A. Ansari et al., Development and validation of HPLC methodfor the determination of S (-) Amlodipine Besylate and its relatedsubstance in tablet formulation by using chiral separation [8]. A simple,specific and sensitive high-performance liquid chromatographic(HPLC) method for determination of S (-) Amlodipine Besylate (SAB)and its related substance i.e. R (+) Amlodipine Besylate (RAB) intablet formulation was developed and validated. Isocratic elution at aflow rate of 1.0 ml/min was employed on chiral column Ultron- ES-OVM, (250 x 4.6 mm, 5 µm). The mobile phase consisted of (Buffer:ACN) (78:22, v/v) in which buffer is mixture of 0.01M disodiumhydrogen phosphate dihydrate and 0.01 M potassium dihydrogenphosphate. The UV detection wavelength was 237 nm and 20 µL ofsample was injected.

The S (-) isomer of amlodipine is found to possess greaterpharmacological effects than R (+) amlodipine. S (-) amlodipine is1000 times more potent than the R (+) isomer in binding to thedihydropyridine receptor. The elution order was found to be for relatedsubstance i.e. RAB (RT = 6.12 min), SAB (RT= 6.93 min, resolution =1.43) at optimized chromatographic condition. The proposed methodcan be utilized for routine analysis and quality control of SAB andRAB in tablet formulation.

Figure 6. Chromatogram obtained from the analysis of SAB andRAB showing good resolution

Anticancer

Nataraj K S et al., Stability indicating analytical method developmentand validation for related substances for Letrozole tablets by RP-HPLC [9]. As per the USP method the test concentration is 10ppm, toolow to detect the impurities as per ICH guidelines and recommendedchanging the test concentration to 100ppm. However, the testconcentration was optimized at 200ppm. Literature reveals thatLetrozole can be analyzed by HPLC using electrochemical,fluorescence, mass spectrometry and UV for detection in bulk materialand pharmaceutical forms. USP method employs amperometricelectrochemical detection. Therefore, in proposed project a successfulattempt has been made to develop simple, accurate and economicmethods for analysis of related substances of Letrozole tablets andvalidated.

Method was developed in reverse phase HPLC mode using, Zodiac

NN

N

NC CN

N

N N

NC CN

Letrozole Letrozole Related Compound A (USP 29)

OH

NC CN

O

NC CN

Impurity A Impurity B

NC CN

O

NC CN

S

CH3

O2

Impurity C Impurity D

Figure 7. Typical Chromatogram of USP method

Figure 8. Typical Chromatogram of modified method

SIL 120- 5-C18 (125*4.6mm, 5.0µm) column with Mobile phase acontaining Filtered and degassed Milli- Q water and mobile phase B-filtered and degassed Milli-Q water and acetonitrile in the ratio of30:70. Forced degradation of the sample was done to determine theintrinsic stability of the drug and to assess the stability of thedeveloped method as per ICH guidelines. Different stress conditionare carried out such as acid degradation, base degradation, peroxidedegradation, water degradation, UV degradation, thermal degradation,sunlight degradation, humidity degradation to generate impurity inthe tablet formulation.



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L. Maheswara Reddy et al., A simple RP-HPLC method for related The column selected was a 5 mm BDS Hypersil C18 (150-4.6 mm)column: an equivalent Phenomenex column has also been used suc-cessfully. The UV detector was set at a wavelength of 260 nm with asensitivity of 0.1 a.u.f.s. An injection volume of 10–20 ml was used.The column temperature was maintained at 20–30°C. The flow rateemployed was 1.5 ml /min throughout the run. The run time employedwas typically 30 min. The method has been validated and is demon-strated to be selective and stability indicating. The method can alsobe applied to bulk assay and impurity testing of solid oral dosageforms such as Melphalan (Alkeran®) Tablets.

Figure 10. Typical chromatogram for freshly prepared solution ofMelphalan.

Peak Compound name Relativenumber retention time

1 Dihydroxy melphalan 0.21

2 Phthalic acid 0.28

3 Morpholino derivative 0.34

4 Methoxyhydroxy melphalan 0.35

5 Chloroethylamino melphalan 0.44

N Ar-

OH

OH

O

O H

O

O H

O

N

A r-

N Ar-

H3CO

H3CO

Cl

NH

Ar-

Table 2. melphalan and its related impurity

The separation was achieved on a Kromosil 5µ C18 column (250 · 4.6mm) using a mobile phase that consists of the buffer (4.5 g of dipotas-sium hydrogen phosphate anhydrous and 2.0 g of tetra butyl ammo-nium hydrogen sulphate (TBAHS) in 1000 ml of water) and methanolin the ratio of 900:100 v/v. The flow rate was maintained at 1.0 ml /min.The detection of the constituents was done at 215 nm using a UVdetector. The retention times of imidazol-1-yl-acetic acid andzoledronic acid were 7.2 and 10.2 min respectively. In the performanceof stress/influenced factors, such as acid or base hydrolysis, hydro-gen peroxide oxidation, light and heat on zoledronic acid and its re-lated substances, inimidazol-1-yl-acetic acid determination. The ad-vantage is present method can be performed more effectively even inthe presence of degradation/stress factors, such as UV-light, sunlight and high temperature conditions, except hydrogen peroxide forthe separation and determination of zoledronic acid and its relatedsubstance, Iimidazol-1-yl-acetic acid. The resolution of the zoledronicacid peak in the present method is 3.0 from its adjacent peaks, whichwas more than the reported methods.

substances of zoledronic acid in pharmaceutical products [10]. Themain objective of this study was to develop a novel, simple, economi-cal, selective, sensitive and stable method indicating the use of re-verse phase-high performance liquid chromatography (RP-HPLC)method for the assay of zoledronic acid and its related substances,imidazol-1-yl-acetic acid present in pharmaceutical products using aUV detector.

Figure 9. Chromatogram of zoledronic acid and its impurity

K. Brightman et al., A stability-indicating method for the determina-tion of melphalan and related impurity content by gradient HPLC [11].The British Pharmacopoeia (BP) monograph and United States Phar-macopeia monograph for melphalan tablets involves an isocratic highperformance liquid chromatographic (HPLC) assay procedure, butthere is no limit placed on the related impurity content. Preliminaryexperimental work to identify a number of related impurities com-monly present namely melphalan dimer and possible polymers, aswell as smaller more polar molecules. This work described in thispaper relates to the development and validation of a gradient HPLCprocedure capable of simultaneous determination of melphalan con-tent and related impurities.



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6 Monohydroxy melphalan 0.52

7 Methoxy melphalan 0.79

8 Ethoxy melphalan methyl ester 0.95

9 Melphalan 1.00

10 Chloroethoxy melphalan 1.05

11 3-Chloro analogue of melphalan 1.16

12 Melphalan dimer 1.27

13 Melphalan methyl ester 1.31

14 Melphalan ethyl ester 1.44

N Ar-

Cl

OH

N Ar-

Cl

H3CO

N

H5C2O

Cl ONH2

O

CH3

N

C l

C l O HNH 2

O

N

O

Cl

Ar-

Cl

N

Cl

Cl OHNH2

O

Cl

N

Cl

Cl ONH2

O

N

CH3

Ar-

N

Cl

C l ONH2

O

CH 3

N

Cl

Cl ONH2

O

H 5C 2

R. Suresh Kumar et al., Development and validation of a stability

Trial-1

In each trial the forced degradation samples were injected for separa-tion of all eight impurities and Exemestane. Chromatograms of peakidentification solution, thermal, acid, base and oxidation degradationobtained with each trial The outcome of each trial is :Trial-2: It was observed that the Imp-8 co-eluted with Exemestane.Trial-3: Imp-8, was separated from Exe peak. However it was eluted atthe same RT of Imp-7.Trial-4: All impurities were separated from each other and withExemestane.Trial-5: Result fromtrial-4 encouraged to use a single organic modifier,instead of two.

Hence this trial was conducted with only acetonitrile as an organicmodifier. Imp-1 and Imp-2 were co eluted. These two are major impu-rities in thermal degradation, and their separation is necessary for themethod to become selective. Few attempts were made to modify thegradient program in trial-5, but there was no improvement in the reso-lution between Imp-1 and Imp-2. Based on above, it was concludedthat trial-4 was highly selective for the quantification of impurities,degradants as well as Exemestane.

This method is highly specific for the quantification of degradationproducts and process impurities of Exemestane.

The chromatographic column used was Hypersil BDS, C-18150mm×4.6mmcolumn with 3µm particles of Thermo scientific make.The mobile phase consists of water (solvent A), and methanol (sol-vent B). The separation was achieved by gradient elution. The HPLCgradient was set as: T/%B: 0/30, 35/60, 40/90, 50/90, 52/30, and 60/30.The flow rate of the mobile phase was kept at 1.0 ml/min and thecolumn temperature was maintained at 45 °C and the chromatogramwas monitored at a wavelength of 247 nm. The injection volume was10µl. A mixture of acetonitrile and water (1:1, v/v) was used as diluent.

indicating LC method for the assay and related substances determi-nation of Exemestane, an aromatase inhibitor [12]. In the literature,several LC methods were reported for determination of Exemestane inbiological samples, no stability indicating method was found in litera-ture search for quantification of Exemestane and related impurities.



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Figure 11. Chromatogram of Exemestane and its impurity

Chromatograms of different trials. (A) Peak identification solution(mixture of Imp-1, Imp-2, Imp-3 and Imp-4 with Exe). (B) Acid degrada-tion. (C) Oxidative degradation. (D) Base degradation. (E) Thermaldegradation. 1. Impurity-1; 2. Impurity-2; 3. Impurity-3; 4. Impurity-4;5. Impurity-5 (major in base degradation); 6. Impurity-6 (major in oxi-dative degradation); 7. Impurity-7 (major in acid degradation); 8. Im-purity-8 (second major in oxidative degradation).

G. Srinivas et al., A Validated stability indicating LC method of assayand related substances for Finasteride [13]. In literature spectroscopicmethod reported for determination of Finasteride in tablets and HPLCmethod for determination of Finasteride in human plasma is reported.No stability indicating methods were reported in the literature for thedetermination of Finasteride and its impurities.

Waters Alliance 2695 separation module (Waters corporation, Milford,USA) equipped with 2695 PDA detector (for specificity and forced

NH

OH

H H

CH3

CH3

NHO

CH3

CH3

CH3

NH

OH

H H

CH3

CH3

OCH3

O

NO

H H

CH3

CH3

NHO

CH3

CH3

CH3

NH

OH

H H

CH3

CH3

NHO

CH3

CH3

CH3

Impurity A Impurity B

Impurity C Impurity D

Figure 12. Structure of Finasteride Impurity

Figure 13. Chromatogram of Finasteride and its impurity

degradation studies) with Empower 2 software was used for the analy-sis. The column used was Symmetry C-18 (75mm X 4.6mm, 3.5µ Wa-ters Corporation, Milford, USA). The optimum composition of mobilephase Water: Acetonitrile (64:36v/v).The flow rate was set to 1 mLmin-1, UV detection was carried out at 210 nm and 10ìl injection vol-ume were maintained. All determinations were performed at ambienttemperature (25° C). In optimized chromatographic conditions ofFinasteride, Imp -A, Imp-B, Imp-C and Imp-D were separated withresolution graters than 2, typical retention times were about 5.9, 7.3,7.9, and 8.9, respectively.



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Anti inflammatory

Srinivas Pola et al., Simultaneous determination of zolmitriptan andits related substances in zolmitriptan tablets 5.0mg and 2.5mg [14].Zolmitriptan high affinity to human 5-HT1B and 5-HT1D receptorsleading to cranial blood vessel constriction. The present study wasaimed at the simultaneous determination of Zolmitriptan and its relatedsubstances in Zolmitriptan tablets by reverse phase HPLC method.Zoming Tablets (Zolmitriptan) are innovator tablets of Astra ZenicaUK Limited available in commercial fixed dose 5 mg and 2.5 mg.

Agilent 1200 series HPLC with VWD detector was used. Ezeochromeelite software version 4.0 is used for Data acquisition. The separationwas performed on Xterra RP C18, 250 X 4.6 mm, 5µm column as astationary phase. Mobile phase comprised of ammonium dihydrogenphosphate buffer and adjusted pH to 9.85 with 30% ammonia solu-tion. Injection volume was 15µl and run time was 70 min and flow rate0.8ml/min. The column was maintained at 40°C temperature and theeluent was detected at 225 nm. Method was validation good resultsfor linearity, accuracy and precision of Zolmitriptan and its relatedsubstances in tablet dosage form. The RSD values for all parameterswere found to be less than 10 for impurities, which indicates thevalidity of method and results obtained by this method are in fairagreement.

Figure 14. Reference chromatogram of impurity mixture

Pakhuri Mehta et al., Development and validation of related sub-stances method by HPLC for analysis of naproxen in naproxen tabletformulations [15]. Naproxen [(+)-2-(6-methoxy-2-naphthyl) propionicacid or (NAP), is a non-steroidal anti-inflammatory drug with antiinflammatory, analgesic and antipyretic properties often preferred toacetylsalicylic acid (aspirin) because of its better absorption follow-ing oral administration and fewer adverse effects. There was no HPLCRelated substances method specified for Naproxen API and tabletformulations in United States Pharmacopoeia.

It was performed on a YMC-ODS A Pack (5µ particles size) (250mm ×4.6mm) column using mobile phase containing Acetonitrile and 10mM Ammonium acetate buffer pH 3.8 in ratio 550:450 v/v (pH 3.8adjusted with acetic acid), considering the pKa of Naproxen 4.2, de-cided to select mobile phase pH range near to pKa. The flow rate 0.8ml/min. Detection was performed at 254 nm and a sharp peak wasobtained for Naproxen at a retention time at about 5.9 ± 0.01 min.

OH

CH3

OH3CO

Figure 15. Structure of Naproxen

Figure 16. Chromatogram of Naproxen

Table 3. Content of Related Substances in Naproxen

Ediga Sasi Kiran Goud et al., RP-HPLC determination of relatedsubstances of tapentadol in bulk and pharmaceutical dosage form[16]. Tapentadol is centrally acting analgesic with a dual mode of actionas an agonist of the µ-opioid receptor and a norepinephrine reuptakeinhibitor. Accurate and precise RP – HPLC method for thedetermination of related substances from bulk sample andpharmaceutical dosage form. The detector responses were linear inthe concentration range of 5.06 – 40.46 µg/ml of drug and its relatedsubstances.

Agilent 1200 series with high pressure liquid chromatographicinstrument provided with auto sampler, and VWD & photo diodearray detector, thermostatted column compartment connected withEZ Chrom software connected with a Zodiac C18 column (250 mm x4.6 mm; 5µm). Using a mobile phase mixture of phosphate buffer pH7.0, acetonitrile and methanol in a gradient elution at a flow rate of1.0ml/min at wavelength of 220 nm. The column temperature wasmaintained at 45ºC. The retention time of Tapentadol was found to be14±0.1 min, methoxy impurity was found to be 39.75±0.1 min andalcohol impurity was found to be 30.9±0.1min. It can be concludedthat the proposed HPLC method is sensitive, specific and reproduciblefor the determination of known and unknown related substances inTapentadol and in pharmaceutical dosage form.



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Figure 17. Chromatogram of Tapentadol with impurities in tabletpreparation

Antibacterial

Clindamycin, with a pKa of about 7.6, was poorly retained under mostreversed-phase conditions. Ion-pair reagent had been used in themobile phases of some other methods to overcome this problem.clindamycin and related components would not be partially ionizedwithin this range, which is generally required for a robust separation.The assay utilized a previously unreported set of conditions, includinga gradient ramp and high-pH mobile phases, to effect the separationwithout the use of an ion-pair reagent in the mobile phase. The methodexhibited good selectivity and sensitivity.

The chromatograph consisted of a Summit System from Dionex(Sunnyvale, CA, USA) comprised of a P-680 binary gradient pumpand ASI-100 autosampler, and a Model 2487 variable wavelength UVdetector from Waters (Milford, MA, USA) set at 214 nm. An XTerraRP18 column (4.6mm×100 mm, 3.5µ mparticle size) from Waters within-line pre-filter was used for the separation. Injection volume andflow rate were 15 µL and 1.0 mL/min, respectively. 10mM carbonatebuffer was prepared by dissolving 1.38±0.10 g potassium carbonate(anhydrous) in 1000mL of water; the pH was adjusted to 10.5 usingconcentrated hydrochloric acid. The composition of mobile phase Awas 90:10 carbonate buffer : acetonitrile. The composition of mobilephase B was 20:80 carbonate buffer : acetonitrile. Six to eight milligramsof sodium nitrate was added to each liter of mobile phase B.

Daniel J. Platzer et al., Developed and validated of a gradient HPLCmethod for the determination of clindamycin and related compoundsin a novel tablet formulation [17]. Clindamycin antibiotic for the treatmentof certain Gram-positive bacterial infections. An in-house methodadapted from was found to be unsuitable for use with the newformulation, because triethyl citrate (a tablet excipient) and degradationproduct A (an impurity resulting from exposure of clindamycin to oneof the excipients, formed over time under certain conditions) interferedwith other clindamycin-related impurities. The degradation productand triethyl citrate were either not well resolved from, or co elutedwith, other clindamycin-related impurities.

Figure 18. Expanded chromatography for a mixture of clindamycinand impurities

The chromatographic separations were performed using eitherSupelco ABZ or Waters XTerra column using a mobile phase, water(+2% triethylamine): acetonitrile 90:10 (v/v%), the pH of the aqueousphase being adjusted to 6.0 with phosphoric acid, with the flow rateof mobile phase of 1.5ml/min at 45 °C. The samples were monitored at290 nm. 20µl volume of sample was injected into HPLC system. Flowrate of 2 ml/min. Under hydrolytic conditions two degradation prod-ucts at tR = 5.90 and 6.73min (acid hydrolysis) and tR = 6.05 and

Predrag Djurdjevic et al., Optimization of separation and determina-tion of moxifloxacin and its related substances by RP-HPLC [18].Moxifloxacin is a synthetic antibacterial agent active against Gram-negative and some Gram-positive bacteria. Its pharmaceutical formu-lations involve tablets and infusions (Avelox®, Avalox®, Tovan®,Bayer AG). isolated and structurally characterized four impurities inbulk moxifloxacin: 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[(S,S)-N-methyl-2,8-diazabicyclo(4,3,0)non-8yl]-4-oxo-3-quinolinecarboxylic acid, methyl-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[(S,S)-2,8-diazabicyclo(4,3,0)non-8-yl]-4-oxo-3-quinolinencarboxylate,1-cyclopropyl-6-fluoro-1,4-dihydro-8-hydroxy-7-[(S,S)-2,8-diazobicyclo(4,3,0)non-8-yl]-4-oxo-3-quinoline carboxylicacid, and 1-cyclopropyl-6,7-difluoro-8-hydroxy-4-oxo-1,4-dihydro-3-quinoline carboxylic acid. The drug was also subjected to acid andalkali hydrolysis, oxidation, dry and wet heat treatment and photodegradation. Significant degradation was observed under hydrolyticconditions while under other conditions degradation was milder. Themain degradant, a decarboxylated product was separated by TLC andcharacterized some moxifloxacin impurities by spectrophotometricmethod. No method for separation and determination of syntheticimpurities of moxifloxacin based on chemometric approach was de-scribed in literature. Simultaneous optimization of that many param-eters requires computer oriented chemometric approach in order tosimplify and accelerate the optimization process. In the present studycomputer simulation software DryLab® was used in developing andoptimizing a reverse-phase HPLC separation of moxifloxacin and itsrelated impurities and degradation products of moxifloxacin.



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6.67min (base hydrolysis) are seen. An impurity peak at tR = 8.02 minwas noted in tablets and infusion solution. It was well resolved fromthe standard moxifloxacin (RRT = 1.54). In the present work a RP-HPLC method for the separation of moxifloxacin impurities was devel-oped with the aid of chemometric approach, validated and used fortheir determination in tablets and infusion. Optimization based onDryLab® computer simulations and on statistical response surfacemethodology led to the practically same optimal conditions for sepa-ration.

Figure 19. Chromatogram of ofloxacin (1 µg/ml), moxifloxacin (200µg/ml) and four synthesis-related impurities (ca. 1 µg/ml) underoptimal conditions.

CONCLUSION:Understanding impurity origin, fate and rejection mechanisms allowappropriate methods and acceptance criteria to be established forroutine quality monitoring. Development methods used during impu-rity investigations, but not necessary for routine use, remain avail-able to evaluate the impact of process upsets or proposed changes.These concepts apply to a wide variety of impurities including re-lated substances, residual solvents, metallic catalysts, toxic impuri-ties, and stereochemical impurities. All impurities were identified anqualified as per ICH limit.

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Source of support: Nil, Conflict of interest: None Declared

and its related substances in zolmitriptan tablets 5.0mg and2.5mg, International Journal of Comprehensive Pharmacy,Vol. 03, Issue 08, 2012, 01-07.

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