a novel rp-hplc method for the quantification of … · 2017-10-19 · 47 article available online...

47Article available online through www.ijrrpas.com

Thamma Narendra Kumar. et al I S S N 2249-1236Research Article

VOL 1, ISSUE (2)

INTERNATIONAL JOURNAL OF RESEARCH AND REVIEWS IN PHARMACY AND APPLIEDSCIENCES

A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF IMPURITIES IN

GLUCOSAMINE HYDROCHLORIDE

Thamma Narendra kumar 1, R. Sreenivasulu 2, Raju satya 1 and Useni Reddy Mallu 2 . 1 Genovo Development Services Ltd,

Bommasandra Industrial Estate, Bangalore, India -560099.2 Department of Chemistry, Sri Krishnadevaraya University,

Anantapur, India-515003.

Article Received on: 10.05.2011 Article Accepted on: 03-07-2011

Corresponding Author

ABSTRACT

Objectives: Glucosamine a natural amino monosaccharide and used for the treatment of osteoarthritis. The mainobjective of this research is to develop a simple, accurate RP-HPLC method for the determination of impurities inglucosamine hydrochloride drug substance and drug product.

Methods: Chromatographic separation was achieved by using Kromasil 100-5C18 300 X 4.0 mm, 5 µm column,mobile phase composed of phosphate buffer (0.8 gm of phosphoric acid, 1.0 ml of Triethylamine and 1.2 gm ofOctane sulphonic acid Sodium salt in 1000 ml of HPLC grade water) and acetonitrile in the ratio of 90:10 (v/v).Column temperature maintained at 25°C, 10µL injection volume and run time was 30min. Analytes absorbancewas measuered at 195 nm.

Results/ Conclusion: The developed method was validated as per ICH guidelines with respect to specificity, limitof detection, limit of quantification, precision, linearity, accuracy, robustness and system suitability. Validationresults were found to be satisfactory and the method applicable for bulk and formulation analysis.

Keywords: Glucosamine hydrochloride, Impurities, RP-HPLC method development, amino sugar andosteoarthritis

Name: Dr.Mallu Useni Reddy

Address: Hyderabad

Email: [email protected]

Phone: 09490310239



VOL 1, ISSUE (2)









ABSTRACT






Address: Hyderabad


Phone: 09490310239



VOL 1, ISSUE (2)









ABSTRACT






Address: Hyderabad


Phone: 09490310239

Thamma Narendra Kumar. et al., IJRRPAS, 2011, 1 (2), 47-61


INTRODUCTION

Glucosamine (1-9) is an amino sugar and a prominent precursor in the biochemical synthesis ofglycosylated proteins and lipids. It is absorbed rapidly from the intestines and transported to the connective tissues andhelps in the restoration of damaged joint tissue in osteoarthritis. It has been used for osteoarthritis, back pain, jointpain and glucoma, by itself or in combination with chondroitin sulfate, diacerein. It is one of the most commonly usedsupplementary medicines as non-vitamin, non-mineral and natural product. It may decrease catabolic activity byinhibiting the synthesis of proteolytic enzymes and other substances that contribute to damage of the cartilage matrix.Glucosamine is required for the synthesis of glycoprotein, glycolipids and glycosaminoglycans(mucopolysaccharides). Glucosamine sulphate and methyl sulfonyl methane (MSM) provided better and more rapidimprovement in patients with osteoarthritis than other combinations. Long-term administration of glucosamine sulfatecan give long-term safety from osteoarthritis of the knee. Glucosamine sulfate was significantly better tolerated thanibuprofen, with fewer adverse effects with better osteoarthritis pain relief. Side effects of glucosamine includesnausea, vomiting, diarrhea. Glucosamine is involved in the formation of nails, tendons, skin, eyes, bones, ligamentsand other connective tissues in the body.

Glucosamine have reported methods by chemical and instrumental methods (9-14) no reported method for thequantification of impurities in glucosamine. Figure-1 represents the chemical structure of glucosamine hydrochlorideand its impurities. The main objective of the present work is to develop a single method for the separation of all thefour impurities of Glucosamine hydrochloride. Validation performed as per ICH and FDA guidelines (15-19).

MATERIALS AND METHODS

Chemicals and reagents

Tablets and Standards of glucosamine hydrochloride impurities namely N-Acetyl –D-Glucosamine impurity(99.0%), 5-Hydroxymethyl-2-Furaldehyde impurity (99.3%), Pyrazine impurity (99.2%), 2-Furaldehyde impurity(99.8%), were obtained from Genovo Development Services Limited, Bangalore, India. All solvents were HPLCgrade and Millipore Milli-Q (Millipore, Milford, MA, USA) high purity water was used for this study.

Equipment

Shimadzu UFLC system (Shimadzu, Kyoto, Japan) equiped with pump, auto sampler and variable UV andPDA detectors were used.

Chromatographic conditions

Mobile phase composed of buffer (Dissolved 0.8 gm of phosphoric acid 85 %, 1.0 ml of Triethylamine and1.2 gm of Octane sulphonic acid salt in 1000 ml of HPLC grade water) and Acetonitrile in the ratio of 90:10 (v/v).Kromasil 100-5C18 300 X 4.0 mm, 5 µm column, 0.5 ml/min of flow rate, column temperature was maintained at25oC and absorabance measuered at 195 nm and injection volume was 10 µl. HPLC grade water used as diluent.

Preparation of Diluted Standard:

Weighed accurately each about 25 mg of N-Acetyl-D-Glucosamine, 5-Hydroxymethyl-2-Furaldehyde,Pyrazine and 2-Furaldehyde standards and transferred into a 25 ml volumetric flask, then added 15 ml of diluent,sonicated for 5 minutes to dissolve and made up the volume with diluent. 1.0ml of resulting solution diluted to 50 mlwith diluent and mixed well.



Preparation of sample solutions:

Twenty Glucosamine hydrochloride 1500 mg tablets were weighed, transferred the tablet powder equivalentto 500 mg of glucosamine hydrochloride to a 50 ml volumetric flask, 35 ml of diluent was added, sonicated to about30 minutes with occasional shaking and made up volume with diluent. These solutions were filtered through a 0.45µm pore size Nylon 66 membrane filter.

Calculation: All impurities of Glucosamine Hydrochloride were quantified against to N-Acetyl Glucosamine peak inthe standard solution (glucosamine and N-Acetyl Glucosamine have same response, it is quantified) with thefollowing formula.

% known impurity= IRT x IW x 1 x P x A

IRDS x 25 x TW x L

% known impurity= IRT x NIW x 1 x P x A

NIRDS x 25 x TW x L

% known impurity= UIRT x NIW x 1 x P x A

NIRDS x 25 x TW x L

Where, IRT = Impurity response from test preparation; IRDS = Impurity average response from dilutedstandard preparation; UIRT = Sum or all Glucosamine Hydrochloride impurity responses from test preparation;NIRDS = N-Acetyl Glucosamine impurity average response from dilutes standard preparation; IW = Impurity weighttaken in mg for diluted standard preparation; NIW = Impurity N-Acetyl Glucosamine weight taken in mg for dilutedstandard preparation; P = Potency of impurity in %; A = Average weight of tablet; L = Label claim of tablet and TW =Test weight taken in mg

The Quantitative estimation of the separated individual impurities and total impurities are done by calculatingthe % of individual known impurities and % of individual unknown impurities.

% Total Impurities = (% of all known Impurities + % of total unknown Impurities)

RESULTS AND DISCUSSIONS

METHOD DEVELOPMENT

Optimization:

Glucosamine is an amino sugar and it is UV inactive molecule. N-acetyl D-glucosamine impurity also UVinactive, other impurites are UV actives but for glucosamine and acetyl glucosamine impurity estimation theabsorbance of blank, standard and test solutions were measured at 195nm. Development trials were performed withphosphate buffer and acetonitrile as mobile phase, C8 250mm columns were used but the elution of all peaks weresame renention time. Finally the separation of all impurities and glucosamine was achieved with Kromasil 100-5C18column, phosphate buffer and Acetonitrile. Optimized method system suitability was evaluated with the replicateinjections of the standard solution. Diluent and diluted standard solution chromatograms were represented in figure-2and 3. Interference of the placebo was studied and found that there was no interference with the all impurities andglucosamine. Figure-4 represents the placebo chromatogram. All known impurities were spiked to test sample andcaculated the recovery results of the impurities. Figure-5 reprents the unspiked test sample chromatogram and figure-6represents the impurities spiked test sample chromatogram. System suitability results were tabulated in table-1.



METHOD VALIDATION:

Specificity:

Specificity is the ability of the method to measure the analyte response in the presence of its potentialimpurities. The specificity of the method was carried out and peak purity study evaluated in the presence of impurities.Diluent, placebo and peak purity of all active peaks were evaluated. Figure-7 to 12 represents the peak purity plot ofall peaks in the test chromatogram.

Precision:

Repeatability of test method was evaluated by analyzing six test preparations by spiking test preparations withGLcN hydrochloride impurities, to get 0.2 % of each GLcN hydrochloride impurity with respect to test concentration(i.e., test concentration 10000 µg/ml). The % RSD of area for each impurity was calculated. Intermediate precision ofthe method was evaluated using different analyst and performing the analysis on different days with differentinstruments, different lot of columns. Precision and intermediate precision comparative results were tabulated inTable-1.

Linearity:

Linearity of the method was validated by diluting stock solution to the required concentrations. The solutionswere prepared at six concentration levels from LOQ to 200% of the specification level (LOQ, 25, 50, 100, 150, 200%). The peak area versus concentration data was treated by least-square linear regression analysis. linearity

Calibration plot was obtained over the calibration ranges tested, i.e. LOQ to 200 %. The correlation coefficientobtained was greater than 0.999. The above result shows that an excellent correlation existed between the peak areaand concentration of glucosamine hydrochloride impurities A-D. Overlaid chromatograms of lenearity wererepresented in figure-13, linearity plots were represented in figure-14 and results were represented in Table-2.

Limits of detection (LOD) and quantification (LOQ):

LOD and LOQ for impurities A, B, C, and D, were determined at a signal-to-noise ratio of 3:1 and 10:1,respectively, by injecting a series of blank solutions and average noise was calculated. Precision study was also carriedout at LOQ level by injecting six individual preparations of impurities and calculating the % RSD of the area. Thedetermined limit of detection, limit of quantification and precision at limit of quantitation values for glucosaminehydrochloride impurities A-D were represented in Table-3.

Accuracy:

Recovery experiments were conducted on to determines accuracy of the method. Study was carried out intriplicate injections of six different concentration levels such as LOQconcentration level, 5, 10, 20, 30, 40µg/ml. Thepercentages of recoveries were calculated and found to be accurate (96.3% to 106.3%). The % recovery values forimpurities are represented in Table-4.

Robustness:

To determine the robustness of the method, experimental conditions were deliberately altered and theresolution between glucosamine hydrochloride and its impurities and tailing factor were recorded with slight changes



in the chromatographic condition flow rate, column temperature and mobile phase composition. Table-5 represents therobustness results for all variations.

Solution stability:

Solution stability of method was carried out by leaving both the diluted standard and test solution of sample intightly capped volumetric flasks at room temperature for 24 hours. Content of impurities A, B, C, D were determinedfor every 4 hrs interval up to 24 hours. No significant changes were observed in the content of four impurities duringsolution stability experiments.

CONCLUSION

Method validation results reveals that the developed RP-HPLC method is precise, accurate and rugged for thequantification of impurities in Glucosamine hydrochloride in bulk and drug products. The method can be used foranalysis of Glucosamine hydrochloride in pharmaceutical industry.

ACKNOWLEDGEMENT

The authors wish to thank Mr. N.S.V. Raju and Dr.Raghupathi (CSO, General Manager, GenovoDevelopment services Ltd. (R&D) and the management of Genovo Development Services Limited, Bangalore forsupporting this research work.

Glucosamine hydrochloride

N-Acetyl-D-Glucosamine 5-Hydroxymethyl-2-Furaldehyde

Impurity (Imp-A) Impurity (Imp-B)



Pyrazine Impurity 2-Furaldehyde Impurity

(Imp-C) (Imp-D)

Figure-1: Chemical structure of glucosamine hydrochloride and its four impurities

Figure-2: Blank chromatogram.

Figure-3: Diluted standard chromatogram




(Imp-C) (Imp-D)







(Imp-C) (Imp-D)






Figure-4: Placebo chromatogram

Figure-5: Un-spiked sample preparation.

Figure-6: Spiked sample preparation of Glucosamine HCl and its impurities A, B, C & D.













Figure-7: Peak purity evaluation of Glucosamine hydrochloride and its impurities A, B, C & D.

Figure-8: Purity plot of Glucosamine

Figure-9: Purity plot of N-Acetyl Glucosamine













Figure-10: Purity plot of Pyrazine

Figure-11: Purity plot of 5-Hydroxymethyl-2-Furaldehyd

Figure-12: Purity plot of 2-Furaldehyde













Figure-13: Overlaid Linearity chromatograms

Figure-14: Linearity plots











Table–1: System Suitability Results

Spiked sample system suitability results

Analyte name TailingFactor Plate Count Resolution

Glucosamine 0.9 2741

N-Acetyl-Glucosamine 1.2 6107 4.1

Pyrazine 2.1 5408 10.8

5-Hydroxymethyl-2-Furaldehyde

1.1 21638 5.5

2-Furaldehyde 1.1 23359 11.1

% RSD of impurity responses

Std.Inj.

Active Ingredient

N-Acetyl-Glucosamine Pyrazine

5-Hydroxy methyl-

2-Furaldehyde2-Furaldehyde

1 796436 1337620 1119001 669524

2 798920 1331256 1120802 670366

3 798831 1331379 1120560 670089

4 797078 1330377 1120393 670014

5 797237 1332913 1120430 670287

6 797429 1332366 1121165 671221

Mean 797655 1332652 1120392 670250

%RSD

0.1 0.2 0.1 0.1

% RSD of % impurity

Std. Inj.N-Acetyl-

Glucosamine

Pyrazine5-Hydroxy methyl-


1 0.209 0.195 0.200 0.235

2 0.210 0.194 0.201 0.235

3 0.212 0.194 0.199 0.233



4 0.215 0.195 0.201 0.235

5 0.217 0.195 0.199 0.235

6 0.217 0.196 0.202 0.237

Mean 0.213 0.195 0.200 0.235

%RSD 1.6 0.4 0.6 0.5

Comparison between Precision and Intermediate Precision Results (Response% RSD)

%RSDN-Acetyl-Glucosam

inePyrazine

5-Hydroxy methyl-


Precision 1.6 0.4 0.6 0.5

IntermediatePrecision

0.9 2.0 0.4 1.0

Table–2: Linearity Regression results

Impurity Name N-Acetyl-Glucosamine

5-Hydroxy-

2-FuraldehydePyrazine 2-Furaldehyde

Line

arity

Sol

utio

ns R

espo

nse Level – 1 (LOQ) 527 1422 684 1257

Level – 2 (5 µg/mL) 209990 317633 274973 160574

Level – 3 (10 µg/mL) 393566 624988 539105 317967

Level – 4 (20 µg/mL) 783843 1266157 1088335 634598

Level – 5 (30 µg/mL) 1160661 1907251 1625331 948453

Level – 6 (40 µg/mL) 1615329 2644301 2270572 1343766

Reg

ress

ion

Res

ults Slope 39145.495 61094.601 56043.351 32220.756

Intercept -2193.800 -20202.434 -15939.010 -11382.276

Correlation Coefficient 0.9994 0.9995 0.9994 0.9990

Standard Error 21814.88 33847.71 32581.16 24992.82



Table–3: LOD and LOQ results

Impurity NameImpurity (%) Impurity (µg/mL) Signal to Noise

Ratio%RSD at

LOQPrecisionLOD LOQ LOD LOQ LOD LOQ

N-Acetyl-Glucosamine 0.000039 0.000124 0.0039 0.0124 3.1 10.0 2.4

5-Hydroxy-2-Furaldehyde 0.000026 0.000130 0.0026 0.0130 3.3 10.3 1.0

Pyrazine 0.000032 0.000214 0.0032 0.0214 3.3 10.1 1.7

2-Furaldehyde 0.000105 0.000377 0.0105 0.0377 3.0 10.0 1.4

Table–4: Recovery results

Level

N-Acetyl-Glucosamine

5-Hydroxy-

2-FuraldehydePyrazine 2-Furaldehyde

%Recovery

%

RSD

%Recovery

%

RSD

%Recovery

%

RSD

%Recovery

%

RSD

LOQ 101.6 0.6 100.9 1.2 106.3 2.1 98.6 0.1

25% 99.7 0.8 96.9 0.9 96.3 0.4 98.5 2.1

50% 100.6 1.0 98.1 0.2 96.9 1.5 101.3 2.3

100% 101.5 1.4 102.0 0.3 101.2 1.0 104.4 1.0

150% 96.8 0.5 97.3 0.2 96.5 0.9 98.9 0.8

200% 97.9 0.2 101.8 0.1 102.0 1.0 101.7 0.3

Table–5: Robustness results

Flow Rate Variation (± 0.05 mL/min)

Active NameLow (0.45 L/min) Actual (0.50 mL/min) High (0.55 L/min)

TF %RSD TF %RSD TF %RSD

N-Acetyl-Glucosamine 1.2 0.2 1.2 0.2 1.3 0.1



5-Hydroxy-2-Furaldehyde 1.1 0.1 1.1 0.0 1.1 0.0

Pyrazine 2.2 0.3 2.3 0.6 2.2 0.1

2-Furaldehyde 1.1 0.1 1.1 0.1 1.1 0.1

Acetonitrile Composition Variation (± 10%)

Active NameLow (90%) Actual (100%) High (110%)




Pyrazine 2.4 0.5 2.3 0.3 2.3 1.1

2-Furaldehyde 1.1 0.3 1.1 0.2 1.1 0.6

Column Oven Temperature Variation (± 5°C)

Active NameLow (20°C) Actual (25°C) High (30°C)




Pyrazine 2.2 0.4 2.3 0.6 2.1 0.2

2-Furaldehyde 1.1 0.1 1.1 0.1 1.1 0.1

TF signifies the tailing factor of respective peak; %RSD signifies the % relative standard deviation of respectiveimpurity response from five replicate injections

REFERENCESS

1. Thakral R et al., Role of glucosamine in osteoarthritis, Current Orthopaedics, 2007, 21, 386-389.2. Bruyere O et al., Glucosamine sulphate reduces osteoarthritis progression in post-menopausal women

with knee osteoarthritis: evidence form two 3-year studies, Menopause, 2004, 11, 138-143.3. Christgau S, Henrotin Y, Tanko LB, Rovati LC, Collette J, Bruyere O, Deroisy R and JY Reginster,

Osteoarthritic patients with high cartilage turnover show increased responsiveness to the cartilage protectingeffects of glucosamine sulphate, Clinical Express Rheumatology, 2004, 22, 36-42.

4. Altman RD et al., Commentary: osteoarthritis of the Knee and glucosamine, Osteoarthritis Cartilage, 2006,11, 963-966.

5. Russell AS, Aghazadeh-Habashi A and F Jamali, Active ingredient consistency of commercially availableglucosamine sulfate products, Journal of Rheumatology, 2002, 29, 2407-2409.

6. Towheed TE et al., Glucosamine therapy for treating osteoarthritis, Cochrane DatabaseSystem Review, 12001.

7. Matheson AJ and CM Perry, Glucosamine: a review of its use in the management of osteoarthritis, DrugsAging, 2003, 20, 1041–1060.



8. Persiani S, Rotini R, Trisolino G, Rovati LC, Locatelli M, Paganini D, Aantonioli D and A Roda, Synovialand plasma glucosamine concentrations in osteoarthritis patients following oral crystalline glucosaminesulphate at therapeutic dose, Osteoarthritis Cartilage, 2007, 15, 764–772.

9. Largo R et al., Glucosamine inhibits IL-1beta-induced NF kappa B activation in human osteoarthritischondrocytes, Osteoarthritis Cartilage, 2003, 11, 290–298.

10. Joseph ZZ, Ted W and M Felicia, Determination of Glucosamine in raw materials and dietary supplementscontaining Glucosamine sulfate and/or Glucosamine hydrochloride by High-Performance LiquidChromatography with FMOC-Su Derivatization: collaborative Study, Journal of Association of Analyt ChemInt, 2005, 88, 1048-1058.

11. Pashkova E, Pirogov A, Bendryshev A, Ivanaynen E and O Shpigun, Determination of underivatizedGlucosamine in human plasma by high-performance liquid chromatography with electrochemical detection:Application to pharmacokinetic study, Journal of Pharmaceutical and Biomedical Analysis, 2009, 50, 671-4.

12. Wayne KW, Kathleen G and GB Andrew, Determination of Glucosamine in nutritional supplements byReversed-Phase ion-pairing HPLC, Journal of Liquid Chromatography and Related Technologies, 2000, 23,2861-2871.

13. PastoriniE et al., Development and validation of a HPLC-ES-MS/MS method for the determination ofGlucosamine in human synovial fluid, Journal of Pharmaceutical and Biomedical Analysis, 2009, 50, 1009-14.

14. Useni Reddy Mallu, K Hussain Reddy, Varaprasad Bobbarala and Somasekhar Penumajji, HPLC MethodDevelopment for Glucosamine Sulphate and Diacerein Formulation, Journal of Pharmacy Research, 2010,3(2), 361-363.

15. International Conference on Harmonization, Q2A: Text on Validation of Analytical Procedures, FederalRegister, 1995, 60 (40), 11260–11262.

16. International Conference on Harmonization, Q2B: Validation of Analytical Procedures: Methodology andAvailability, Federal Register, 1997, 62 (96), 27463–27467.

17. FDA, Analytical Procedures and Methods Validation: Chemistry, Manufacturing and ControlsDocumentation, Availability, Federal Register (Notices), 2000, 65(169), 52776–52777.

18. www.fda.gov/cder/guidance/cmc3.pdf19. USP 25–NF 20, Validation of Compendial Methods Section (1225) (United States Pharmacopeal

Convention, Rockville, Maryland, USA, 2002), 2256.

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