Journal of Chromatographic Science Advance Access published November 17, 2013 Journal of Chromatographic Science 2013;1– 11 doi:10.1093/chromsci/bmt163
Article
Validated Stability Indicating RP-HPLC for Quantitation of Nitazoxanide in Presence of Its Alkaline Degradation Products and Their Characterization by HPLC-Tandem Mass Spectrometry Maha Hegazy1*, Amira Kessiba2, Ahmed Emad El Gindy2 and Mohamed Abdelkawy1 1
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, 11562 Cairo, Egypt and 2Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, KM 28 Cairo – Ismailia Road (Ahmed Orabi District), Cairo, Egypt
*Author to whom correspondence should be addressed. Email: [email protected] Received 5 February 2013; revised 14 October 2013
A simple and sensitive stability indicating HPLC method was developed and validated for quantitative determination of Nitazoxanide (NTZ), a new antiprotozoal drug, in presence of degradation products generated under forced alkaline hydrolysis. Chromatographic separation was achieved on Inertsil C8-3 column (150 3 4.6 mm i.d.) using a mobile phase composed of acetonitrile: 50 mM ammonium acetate buffer (50:50, v/v, pH 5.0 adjusted with acetic acid) at a flow rate of 1 mL/min. Quantification was achieved with UV detection at 298 nm based on relative peak area. The method was linear over the concentration range of 0.8–50 mg/mL (r 5 0.9999) with a limit of detection and quantification 0.0410 and 0.1242 mg/mL, respectively. The developed method has the requisite accuracy, selectivity, sensitivity and precision to assay NTZ in presence of its degradation products either in bulk powder or in pharmaceutical formulations. The degradation products were then identified by HPLC-MS/MS analysis using an electrospray ionization source and an ion trap analyzer.
lower sensitivity of these methods as they determined NTZ at higher concentration levels than the present work. The present work focuses on detailed study of the alkaline degradation of NTZ due to its high instability in alkaline medium and our aim was to develop a simple and sensitive stability indicating the RP-HPLC method for the quantitative determination of NTZ in presence of its alkaline degradation products and the method was validated according to ICH guidelines (47). The degradation products were characterized by liquid chromatographytandem mass spectrometry analysis using an electrospray ionization (ESI) source and an ion trap analyzer. Experimental Instruments HPLC instrumentation and conditions Samples were loaded into Rheodyne 7725i injection valve, equipped with a 20-mL sample loop (Rheodyne, Berkeley, CA,
Introduction Nitazoxanide (NTZ), as shown in Figure 1A, is a nitrothiazole benzamide compound that has a wide range of antimicrobial activity against parasites, bacterial and viral pathogens. The broad spectrum of in vivo activity is related to its desacetyl derivative, tizoxanide (Figure 1B), and includes intracellular and extracellular protozoa, helminthes, aerobic and anaerobic bacteria and virus (1 –8). It is chemically designated as N-(5-nitro-2-thiazolyl) salicyl amide acetate (9). It is prescribed for treatment of diseases caused by Giardia intestinalis and Cryptosporidium in immune-compromised patients, including those with AIDS or HIV infection. The drug represents a significant advance in the treatment of intestinal parasitical infections worldwide (10, 11). NTZ is not an official drug. Several chromatographic (12 –31), spectrophotometric (32 –45) and electrochemical (46) analytical methods have been reported for the determination of NTZ in bulk powder, pharmaceutical formulations alone or in combination with other drugs and/or in biological fluids. Several stability indicating HPLC methods (13– 15, 22) have been reported for the determination of NTZ in the presence of its degradation products. These methods showed that NTZ undergoes slight degradation when exposed to acid, oxidative and photodegradation, but it is highly unstable in alkaline medium and is stable to heat. None of these methods provided identification of the degradation products formed as well as the
Figure 1. The chemical structure of Nitazoxanide (NTZ) (A). The chemical structure of tizoxanide (B).
# The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
USA). HPLC separation and quantitation were made on Inertsil C8-3 column (150 4.6 mm i.d., 5 mm particle size) with a mobile phase consisting of acetonitrile: 50 mM ammonium acetate buffer (50:50, v/v), pH was adjusted to 5.0 using acetic
acid. The mobile phase was filtered using 0.45 mm membrane filters (Millipore, Milford, MA, USA) and degassed by ultrasonic vibrations for 15 min prior to use. An isocratic pump was used to deliver the mobile phase at a flow rate of 1 mL/min (Agilent 1100
Figure 2. Typical HPLC chromatograms of (A) pure NTZ 10 mg/mL (tR ¼ 5.295), (B) alkaline degradation products formed after 15 min reflux with 0.1 N NaOH (NTZ DegI) equivalent to 10 mg/mL (tR ¼ 1.882, 2.191, 2.635), (C) alkaline degradation products formed after 1 h reflux with 0.1 N NaOH (NTZ DegII) equivalent to 10 mg/mL (tR ¼ 1.882, 2.639).
2 Hegazy et al.
Series Iso pump G1310A, Agilent Technologies, Santa Clara, CA, USA). The samples were filtered using 0.45 mm membrane filters. The UV–Vis detector (Agilent 1100 Series VWD G1314A) was set at 298 nm. Data acquisition were performed on Agilent LC ChemStation software. All determinations were performed at ambient temperature (258C). Liquid chromatography–tandem mass spectrometry instrumentation and conditions Liquid chromatography-tandem mass spectrometry (LC–MS/MS) composed of Applied Biosystem 3200 Q trap with LC consisting of a solvent delivery system and an autosampler (Agilent 1200). The mobile phase consisted of acetonitrile:10 mM ammonium acetate buffer (pH ¼ 4.5) in the ratio of 50:50 at a flow rate 1 mL/min. The mobile phase containing the analytes was flowing directly into a positive and negative electrospray ionization probe (+ESI)
of a tandem triple quadruple mass spectrometer (Q trap). The instrument permits various scanning modes such as MS, MS/MS. Tuning parameters for MS scan for NTZ were optimized in negative mode by direct infusion of NTZ solution at a concentration of 1 mg/mL. The capillary voltage was adjusted at 4 kV, collision energy at 13 eV and collision gas pressure at 3 mbar. All data acquisition were controlled by Analyst 1.5 software.
Materials Pure standard NTZ standard material was supplied by Sigma Pharmaceutical Industries, 1st zone, Mubarak industrial zone, Quwasnah, El Monofeya-Egypt, it was certified to contain 99.5% (w/w) according to the manufacturer’s method.
Figure 3. HPLC chromatograms of (A) resolved mixture of 10 mg/mL of NTZ (tR ¼ 5.302), and 10 mg/mL of its alkaline degradate (NTZ DegI) (tR ¼ 1.879, 2.188, 2.633), (B) resolved mixture of 10 mg/mL of NTZ (tR ¼ 5.299), and 10 mg/mL of its alkaline degradate (NTZ DegII) (tR ¼ 1.879, 2.634) mobile phase is acetonitrile: 50 mM ammonium acetate buffer (50:50, v/v, pH adjusted to 5.0 using acetic acid).
Validated Stability Indicating RP-HPLC Method 3
Pharmaceutical formulations (i) Nanazoxid w tablets (Batch No. 10206) are labeled to contain 500 mg of NTZ per tablet, produced by Medizen Pharmaceuticals, Borg El arab-Alex.-Egypt for Utopia Pharmaceuticals. (ii) Nitazode w suspension (Batch No. 02785) is labeled to contain 100 mg of NTZ per 5 mL suspension, manufactured by Sigma Pharmaceutical Industries for Al Andalous Medical Co., Cairo, Egypt. Chemicals and reagents All chemicals and reagents used throughout this work were of analytical grade and solvents were of HPLC grade: (i) Sodium hydroxide (Adwic, Egypt) (ii) Ammonium acetate (Adwic, Egypt) (iii) Acetic acid (Adwic, Egypt). (iv) Hydrochloric acid (Sigma-Aldrich, Germany). (v) Acetonitrile (Sigma-Aldrich, Germany). Solutions NTZ stock standard solution NTZ standard solution (100 mg/mL) was prepared by accurately weighing 10 mg of NTZ into 100 mL volumetric flask, dissolve in and complete to volume with acetonitrile. The solution was protected from light by wrapping the flask with aluminum foil. Alkaline degradation products stock solution An accurately weighed amount of pure NTZ (10 mg) was transferred into two separate conical flasks, the first one was refluxed with 20 mL of 0.1 N NaOH for 15 min (NTZ DegI) and the second one was refluxed with 20 mL of 0.1 N NaOH for 1 h (NTZ DegII). The solutions were then neutralized by adjusting the pH using 0.1 N HCl and transferred quantitatively to 100 mL volumetric flasks. The volumes were then completed using acetonitrile to produce concentration equivalent to 100 mg/mL of each. The degradation process was followed every 15 min and complete degradation was confirmed by HPLC using 50 mM ammonium acetate buffer: acetonitrile (50:50, v/v, and the pH was adjusted to 5.0 using acetic acid). Laboratory prepared mixtures Aliquots (1.8–0.2 mL) of NTZ standard solution (100 mg/mL) equivalent to 18 –2 mg/mL were accurately transferred into two series of 10 mL volumetric flasks, to the first series aliquots
Table II Accuracy Results of the Proposed HPLC Method for the Determination of NTZ Pure Samples NTZ (mg/mL)
Recovery %
Taken
Found
2 5 8 15 18 25 35 45 Mean RSD%
2.01 5.02 7.99 14.91 17.96 25.11 34.96 45.15
100.5 100.4 99.9 99.4 99.8 100.4 99.9 100.3 100.1 0.397
Table III Determination of NTZ in Presence of Its Alkaline Degradation Products (NTZ DegI and NTZ DegII) in Laboratory Prepared Mixtures by the Proposed HPLC Method Mixture no.
NTZ DegI %
NTZ (mg/mL) Taken
Found
Recovery %
1 2 3
10 50 90
18 10 2
17.72 10.05 2.01
98.4 100.5 100.5
Mean RSD%
99.8 1.192
NTZ DegII % 10 50 90
NTZ (mg/mL) Taken
Found
Recovery %
18 10 2
17.79 9.97 2.03
98.8 99.7 101.5
Mean RSD%
100.0 1.362
Table IV Results Obtained by Applying the Proposed HPLC Method for the Determination of NTZ in Nanazoxidw Tablets and Nitazodew Suspension and Results Obtained by Applying the Standard Addition Technique Product
Found*% + RSD%
w
Nanazoxid tablets 500 mg NTZ/tablet B.N.10206
99.1 + 0.497
Nitazodew suspension 100 mg NTZ/5 mL B.N.02785
98.9 + 1.151
Standard addition technique Added (mg/mL)
Recovery%
18 20 22 Mean 100.6 RSD% 0.667 18 ‘ 22 Mean 99.1 RSD% 0.546
100.0 100.4 101.3
98.5 99.5 99.4
*Average of four determinations.
Table I Validation Parameters for the Determination of Pure NTZ Samples by the Proposed HPLC Method Parameter
NTZ
Range Slope Intercept SE of the slope SE of the intercept Correlation coefficient (r) LOD LOQ Precision Repeatability (%) Intermediate precision (%)
0.8 –50 mg/mL 0.1009 0.0079 0.0003 0.0102 1 0.0410 0.1242
4 Hegazy et al.
0.713 0.787
Table V Statistical Comparison of the Results Obtained by the Proposed HPLC Method and the Reported HPLC Method(B) for the Analysis of NTZ in Pure Form Parameter
NTZ
Reported HPLC method
Mean SD n Variance Student’s t F
100.1 0.397 8 0.158 0.548 (2.447)* 2.273 (4.120)*
100.2 0.598 5 0.358
*The values between parentheses are the tabulated values for t and F at p ¼ 0.05.
(0.2 –1.8 mL) of NTZ DegI stock solution (100 mg/mL) were accurately added and to the second one, similar aliquots of NTZ DegII stock solution (100 mg/mL) equivalent to 2 –18 mg/mL were accurately added. The volumes were then completed with the mobile phase to prepare mixtures containing from 10 to 90% of NTZ DegI and NTZ DegII. Solutions for LC –ESI –MS/MS analysis An aliquot of each of NTZ standard solution and NTZ degradation products (NTZ DegI, NTZ DegII) stock solutions was diluted with mobile phase to give a final concentration of 1 mg/ mL. The prepared samples were injected into LC –MS using positive and negativeESI. The MS and MS/MS scans were selected for the identity studies.
Procedure Calibration of standard solutions Different aliquots (0.08–5 mL) of NTZ standard solution (100 mg/mL), equivalent to 0.8– 50 mg/mL, were transferred
into a series of 10 mL volumetric flasks and the volumes were adjusted with the mobile phase. An aliquot of 20 mL of each solution was injected into the chromatographic system and processed according to the previously described conditions.
Application to pharmaceutical formulations Nanazoxidw tablets. Six Nanazoxid tablets (labeled to contain 500 mg NTZ) were accurately weighed and finely powdered. An accurate weight of the powdered tablets equivalent to 10 mg NTZ was transferred into a 100-mL volumetric flask, extracted with 50 mL acetonitrile in an ultrasonic bath for 10 min and diluted to volume with the same solvent. The solution (100 mg/mL) was shaken again for 10 min in the ultrasonic bath and filtered. Different aliquots (0.4, 0.6 and 1 mL) were transferred into a series of 10 mL volumetric flasks and the volumes were completed with the mobile phase to produce solutions of concentrations equivalent to 4, 6 and 10 mg/mL of NTZ, respectively. The procedure was completed as described under calibration and the concentrations of NTZ were calculated from the corresponding regression equation.
Figure 4. (A) MS spectrum of NTZ solution corresponding to the peak at tR ¼ 5.29 in Figure 2A), (B) MS/MS product ion spectrum of m/z 305.9 corresponding to NTZ.
Validated Stability Indicating RP-HPLC Method 5
Nitazodew suspension. The whole bottle was emptied and the powder was accurately weighed. An accurately weighed amount of powder equivalent to 10 mg NTZ was transferred into a 100-mL volumetric flask, then the procedure was completed as described above.
Results and discussion The literature survey revealed that NTZ is liable to acid, alkaline, oxidative and photodegradation (12– 15, 22, 24). In this work, we are focusing on the alkaline degradation of NTZ as it shows a high degree of instability in alkaline medium.
HPLC analysis Nitazoxanide was subjected to hydrolysis under alkaline-stress conditions by refluxing with 0.1 N NaOH and degradation was followed every 15 min by HPLC [C18 column, mobile phase: 50 mM ammonium acetate buffer– acetonitrile (50:50, v/v), pH adjusted to 5.0 using acetic acid and detection at 298 nm]. It was found that complete degradation occurred after 15 min (NTZ
DegI) with the formation of three degradation products. By following the degradation procedure, it was found that the peak area corresponding to one of the formed degradates decreased gradually with the increase in the area of one of the other two peaks. After 1 h (NTZ DegII), two final degradation products were obtained without further change. Representative chromatograms showing the degradation process are shown in Figure 2. In order to optimize the proposed HPLC method, several trials were carried out to obtain good and optimum separation of NTZ from its degradation products. Initially, methanol and acetate buffer in different ratios as (60:40 and 65:35, v/v) were tried, but this showed peak broadening. Acetonitrile was tried with methanol and acetate buffer in the ratio (30:30:40, v/v/v), but this resulted in decreased peak symmetry, so methanol was totally replaced by acetonitrile. The effect of changing the ratio of the organic modifier on the retention times was investigated and finally, it was found that acetonitrile:acetate buffer in the ratio (50:50, v/v, pH adjusted to 5.0 using acetic acid) with a flow rate of 1 mL/min was most suitable to get resolved and sharp peaks. The optimum wavelength for detection and quantification was 298 nm, at which good detector response was obtained with symmetrical peaks.
Figure 5. MS spectra of (A) NTZ alkaline degradate (NTZ DegI) corresponding to the peaks at tR ¼ 1.882 (m/z 136.9) and tR ¼ 2.191 (m/z 178.9) in Figure 2B in negative ion mode, (B) NTZ DegI corresponding to the peak at tR ¼ 2.635 in Figure 2B in positive ion mode. 6 Hegazy et al.
Figure 6. MS/MS product ion spectra of (A) m/z 178.9 in negative mode, (B) m/z 136.9 in negative mode, (C) m/z 146.1 in positive mode.
Validated Stability Indicating RP-HPLC Method 7
Upon applying the previously described HPLC optimum experimental conditions, good and efficient separation was observed between NTZ and its alkaline degradation products (NTZ DegI and NTZ DegII), Figure 3. Linear relationship was obtained for NTZ between the relative peak areas and the corresponding concentrations, Supplementary data, Figure S1. The regression equation was computed and found to be: A ¼ 0:1008C þ 0:0067; r ¼ 0:9999 where A is the relative peak area, C is NTZ concentration in mg/mL and r is the correlation coefficient.
System suitability was checked by calculating different parameters such as capacity factor, tailing factor, column efficiency (N), selectivity and resolution factors, where the system was found to be suitable relative to the reference values, Supplementary data, Table S1. Validation (47) of the proposed method was constructed by determining the linearity, range, accuracy and precision (Table I). The proposed HPLC method was successfully applied for the determination of NTZ in pure powdered form with mean percentage recoveries of 100.1 + 0.397% (Table II).
Figure 7. MS spectra of (A) NTZ alkaline degradate (NTZ DegII) corresponding to the peak at tR ¼ 1.882 in Figure 2C in negative mode, (B): NTZ DegII corresponding to the peak at tR ¼ 2.639 in Figure 2C in positive mode.
8 Hegazy et al.
The specificity of the proposed method was proved by the analysis of laboratory prepared mixtures containing different ratios of NTZ-NTZ DegI and NTZ-NTZ DegII, satisfactory results were obtained for NTZ in the presence of up to 90% of both NTZ DegI and NTZ DegII (Table III). The proposed HPLC method has been successfully applied for the determination of NTZ in Nanazoxidw tablets and Nitazodew suspension. The validity of the method was further assessed by applying the standard addition technique (Table IV). The results obtained for the analysis of NTZ in its pure form by the proposed HPLC method were statistically compared with those obtained by applying a reported HPLC method (14). The calculated t and F values were less than the tabulated ones at 95% confidence level, which reveals that there is no significant difference between the two methods with respect to accuracy and precision (Table V).
Identification of degradation products by LC– ESI–MS/MS analysis The MS scan of a reference solution of NTZ in negative ion mode showed a strong signal at m/z 305.9 [M2H]2 which refers to the deprotonated molecular (parent) ion of the drug (Figure 4A). The MS/MS scan of NTZ showed daughter ions at m/z (305.9, 263.9 and 185.9) (Figure 4B), the signal formed at m/z 263.9 [M2H]2 was assigned to tizoxanide (Figure 1B), which is the active metabolite of NTZ formed through de-acetylation of the parent ion. Scanning of NTZ DegI solution in negative ion mode showed two strong signals at m/z 178.9 [M2H]2 and 136.9 [M2H]2 (Figure 5A), which were assigned to acetyl salicylic acid and salicylic acid, respectively. By scanning NTZ DegI solution in positive ion mode, a signal appeared at m/z 146.1 [M2H]þ (Figure 5B), which corresponds to 5-nitro-1,3-thiazol-2-amine resulting from the hydrolysis of the amide linkage of NTZ. The MS/MS scan of
Figure 8. Schematic diagram showing the suggested NTZ degradation products.
Validated Stability Indicating RP-HPLC Method 9
the product ion at m/z 178.9 [M2H]2 corresponding to acetylsalicylic acid showed a major fragment in negative mode at m/z 135.9 [M2H]2 resulting from the loss of the acetyl group (Figure 6A). The ion at m/z 136.9 [M2H]2 corresponding to salicylic acid shows a major fragment at m/z 93 [M2H]2 in the MS/ MS scan in negative ion mode (Figure 6B). By fragmentation of the ion at m/z 146.1 [M2H]þ in positive mode, it showed a major fragment at m/z 105 [M2H]þ in the MS/MS spectrum (Figure 6C). Scanning NTZ DegII solution in negative ion mode showed a strong signal at m/z 136.9 [M2H]2 (Figure 7A), which was assigned to salicylic acid as described above, and in positive ion mode, it showed a strong signal at m/z 146.1 [M2H]þ (Figure 7B), corresponding to 5-nitro-1,3-thiazol-2-amine. Tuning parameters for MS and MS/MS scans of NTZ and its alkaline degradation products are listed in Supplementary data, Table S2. The degradation pathway of NTZ and the structures of the degradation products formed are shown in Figure 8. It was found that the main degradation route of NTZ is via hydrolysis of the amide linkage and the ester linkage. Upon refluxing of NTZ with 0.1 N NaOH for 15 min (NTZ DegI), complete degradation occurred through hydrolysis of the amide linkage and partial hydrolysis of the ester linkage giving three degradation products which are acetyl salicylic acid, salicylic acid and 5-nitro-1,3thiazol-2-amine. By continuing the degradation procedure, gradual de-acetylation of the formed acetyl salicylic acid occurred giving salicylic acid, which explains the gradual decrease in the peak area of one of the degradation products with the increase in the area of the other one in HPLC. After 1 h, only two degradation products were detected, which are salicylic acid and 5-nitro-1,3-thiazol-2-amine. So NTZ must be properly stored to avoid the presence of salicylic acid resulting from the degradation process, which exposes the patient to toxicity.
Conclusion The suggested method is simple, accurate, selective and sensitive. Application of the proposed method to the analysis of NTZ in laboratory prepared mixtures and pharmaceutical formulations showed that neither the degradation products nor the excipients interfere with the determination, indicating that the proposed method could be applied as stability indicating method for the determination of NTZ in presence of its degradation products either in bulk powder or in pharmaceutical formulations. Also identification of the degradation products was successfully achieved by LC –ESI –MS/MS analysis.
Supplementary data Supplementary data are available at Journal of Chromatographic Science online. References 1. Dubreuil, L., Houcke, I., Mouton, Y., Rossignol, J.F.; In vitro evaluation of activities of nitazoxanide and tizoxanide against anaerobes and aerobic organisms; Journal of Antimicrobial Agents and Chemotherapy, (1996); 40: 2266–2270.
10 Hegazy et al.
2. Cabello, R.R., Guerrero, L.R., Garcia, M.R.M., Cruz, A.G.; Nitazoxanide for the treatment of intestinal protozoan and helminthic infections in Mexico; Journal of Transactions of the Royal Society of Tropical Medicine and Hygiene, (1997); 91: 701–703. 3. Megraud, F., Occhialini, A., Rossignol, J.F.; Nitazoxanide a potential drug for eradication of Helicobacter pylori with no cross-resistance to metronidazole; Journal of Antimicrobial Agents and Chemotherapy, (1998); 42: 2836–2840. 4. Rossignol, J.F., Ayoub, A., Ayers, M.S.; Treatment of diarrhea caused by Cryptosporidium parvum: a prospective randomized, double-blind, placebo-controlled study of nitazoxanide; Journal of Infectious Diseases, (2001); 184: 103–106. 5. Rossignol, J.F., Ayoub, A., Ayers, M.S.; Treatment of diarrhea caused by Giardia intestinalis and Entamoeba histolytica or Entamoeba dispar: a randomized, double-blind, placebo-controlled study of nitazoxanide; Journal of Infectious Diseases, (2001); 184: 381–384. 6. Rossignol, J.F., Abou Zekry, M., Hussein, A., Santoro, M.G.; Effect of Nitazoxanide in treating severe Rotavirus diarrhea: a randomized, double-blind, placebo-controlled trial; Lancet, (2006); 368: 124– 129. 7. Esposito, M., Mu¨ller, N., Hemphill, A.; Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum; International Journal of Parasitology, (2007); 37: 183– 190. 8. Korba, B.E., Montero, A.B., Farrar, K., Gaye, K., Mukerjee, S., Ayers, M.S., et al.; Nitazoxanide, tizoxanide and other thiazolides are potent inhibitors of hepatitis B virus and hepatitis C virus replication; Journal of Antiviral Research, (2008); 77: 56– 63. 9. Budavaried, S. The Merck Index, an Encyclopedia of Chemicals, Drugs and Biologicals. (14th ed.). Merck & Co., Inc., Whitehouse Station, NJ, USA, (2006). 10. Sweetman, S.C. Martindale: The Complete Drug Reference. (33rd ed.) The Pharmaceutical Press, London, (2002), pp. 598– 599. 11. Stockis, A., Deroubaix, X., Lins, R., Jeanbaptiste, B., Calderon, P., Rossignol, J.F.; Pharmacokinetics of nitazoxanide after single oral dose administration in 6 healthy volunteers; International Journal of Clinical Pharmacology and Therapeutics, (1996); 34: 349–351. 12. Gopu, C.L., Thomas, S., Paradkar, A.R., Mahadik, K.R.; A validated stability indicating HPTLC method for determination of nitazoxanide; Journal of Scientific & Industrial Research, (2007); 66: 141–145. 13. Jadhav, A.S., Pathare, D.B., Shingare, M.S.; A validated stability indicating RP-LC method for nitazoxanide, a new antiparasitic compound; Chromatographia, (2007); 66: 595– 600. 14. Malesuik, M.D., Cardoso, S.G., Steppe, M.; Development of a validated stability indicating LC method for nitazoxanide in pharmaceutical formulations; Chromatographia, (2008); 67: 131–136. 15. Rane, V.P., Sangshetti, J.N., Patil, K.R., Yeole, R.D., Shinde, D.B.; Stability indicating LC determination of nitazoxanide in bulk drug and in pharmaceutical dosage form; Chromatographia, (2008); 67: 455– 459. 16. Sakamoto, T., Hiyama, Y.; Rapid determination of nitazoxanide in tablets using reversed-phase ultra-performance liquid chromatography and high performance liquid chromatography; Die Pharmazie, (2008); 63: 503–507. 17. Kalta, R.R., Sharma, R., Chaturvedi, S.C.; Simultaneous RP-HPLC determination of nitazoxanide and ofloxacin in combined tablet dosage form; Indian Journal of Pharmaceutical Sciences, (2008); 70: 491–494. 18. Zhao, Z., Zhang, L., Xue, F., Wang, X., Zheng, W., Zhang, T., et al.; Liquid chromatography-tandem mass spectrometry analysis of nitazoxanide and its major metabolites in goat; Journal of Chromatography B, (2008); 875: 427–436. 19. Jadhav, V.Y., Gandhi, S.V., Dhavale, N.D., Sabnis, S.S.; RP-HPLC determination of nitazoxanide in bulk and different tablet formulations; Eurasian Journal of Analytical Chemistry, (2008); 3: 404–409. 20. Kumar, R.S., Nallasivan, P.K., Saravanakumar, S., Kandasamy, C.S., Venkatnarayanan, R.; Simultaneous RP-HPLC estimation of nitazoxanide and ofloxacin in tablet dosage forms; Asian Journal Research Chemistry, (2009); 2: 43 –45. 21. Ruiz-Olmedo, M.I., Gallegos-Perez, J.L., Calderon-Gonzalez, K.G., Franco-Perez, J., Jung-Cook, H.; Sensitive high performance liquid
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
chromatographic assay for nitazoxanide metabolite in plasma; Die Pharmazie, (2009); 64: 419–422. Malesuik, M.D., Goncalves, H.M.L., Paim, C.S., Schapoval, E.E.S., Steppe, M.; LC: analysis of photodegradation kinetics of nitazoxanide in pharmaceutical formulations; Journal of Chromatographic Science, (2009); 47: 745– 748. Vipul, P.R., Devanand, B.S.; Simultaneous high performance liquid chromatographic determination of nitazoxanide and ofloxacin in tablet formulation; Eurasian Journal of Analytical Chemistry, (2009); 4: 304–310. Malesuik, M.D., Goncalves, H.M.L., Paim, C.S., Jablonski, A., Schapoval, E.E.S., Steppe, M.; Stability indicating CZE method and stress degradation studies of nitazoxanide; Journal of Liquid Chromatography and Related Technologies, (2010); 33: 375–389. Valarmathy, J., Samueljoshua, L., Rathinavel, G., Senthilkumar, K.L.; Method development and validation for assay of nitazoxanide in tablet using RP-HPLC; International Journal of ChemTech Research, (2010); 2: 1263–1266. Premanand, D.C., Senthilkumar, K.L., Senthilkumar, B., Saravanakumar, M., Thirumurthy, R.; A validated RP-HPLC method for simultaneous estimation of nitazoxanide and ofloxacin in pharmaceutical formulation; Der Chemica Sinica, (2010); 1: 1– 5. Singh, H.P., Agal, S.K., Sharma, C.S., Ranawat, M.S., Ankalgi, A.D.; RP-HPLC methods for estimation of nitazoxanide single and simultaneous estimation of nitazoxanide with ofloxacin in pharmaceutical dosage forms; Journal of Chemical and Pharmaceutical Research, (2010); 2: 984–992. Sharma, S., Bhandari, A., Choudhary, V.R., Rajpurohit, H., Khandelwal, P.; RP-HPLC method for simultaneous estimation of nitazoxanide and ofloxacin in tablets; Indian Journal of Pharmaceutical Sciences, (2011); 73: 84 –88. Bhale, S., Patankar, R.D.; RP-HPLC development and validation of nitazoxanide in tablet dosage form; International Journal of Pharmacy and Pharmaceutical Sciences, (2011); 3: 71– 73. Sharma, M.C., Sharma, S.; Development and validation of a dissolution method with isocratic high performance liquid chromatographic determination of nitazoxanide and ofloxacin in pharmaceutical dosage form; American-Eurasian Journal of Scientific Research, (2011); 6: 182–187. Mehta, D., Seth, A.K., Kumar, S., Yadav, Y.C.; HPLC method for simultaneous estimation of ofloxacin and nitazoxanide in its tablet dosage form; Pharma Science Monitor, (2011); 2: 1 –12. Kapse, G.K., Prabhakar, G., Raju, S.A.; Spectrophotometric methods for the estimation of nitazazoxanide in pharmaceutical formulations; Indian Journal of Pharmaceutical Sciences, (2006); 68: 403– 406. Lakshminarayana, K.V., Manohara, Y.N., Gurupadayya, B.M.; Development and validation of spectrophotometric methods for the estimation of nitazoxanide in tablet dosage forms; Indian Journal of Pharmaceutical Sciences, (2007); 69: 147–149. Senthilraja, M.; Simultaneous UV spectrophotometric method for the estimation of nitazoxanide and ofloxacin in combined dosage form;
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
Research Journal of Pharmacy and Technology, (2008); 1: 469– 471. Vanitha, J., Kumar, A., Ganesh, M., Vetrichelvan, T.; Estimation of nitazoxanide by UV spectrophotometric method; Journal of Global Pharma Technology, (2009); 1: 51 –53. EL-Desoky, H.S., Ghoneim, M.M., Abdel-Galeil, M.M.; A direct spectrophotometric method for determination of antiparasitic drug nitazoxanide in a bulk form and pharmaceutical formulation; Chemia Analityczna, (2009); 54: 1065–1077. Sherje, A.P., Chokshi, J., Satam, C.A., Chaudhary, D., Vanshiv, S.D.; Spectrophotometric determination of nitazoxanide and ofloxacin in combined dosage form; Journal of Pharmacy Research, (2010); 3: 72– 74. Malesuik, M.D., Paim, C.S., Schapoval, E.E.S., Steppe, M.; Development of a simple, rapid and validated spectrophotometric method for nitazoxanide in pharmaceutical formulations and comparison with HPLC; Quimica Nova, (2010); 33: 739–742. Bindaiya, S., Bankey, S., Jain, D.; Simultaneous determination of nitazoxanide and ofloxacin in tablet by ultraviolet spectrophotometry (dual wavelength method); International Journal of ChemTech Research, (2010); 2: 11– 15. Sherje, A.P., Anand, R., Sriram, W., Vanshiv, S.D.; Mixed hydrotropy in spectrophotometric analysis of nitazoxanide; International Journal of ChemTech Research, (2010); 2: 1966–1969. Adhikari, L., Sahu, S., Jagdev, S.; Development and validation of spectrophotometric and colorimetric method for the determination of nitazoxanide in its bulk and pharmaceutical dosage form; International Journal of ChemTech Research, (2011); 3: 131– 135. Singh, H.P., Sharma, C.S., Ankalgi, A.D., Agal, S.K., Ranawat, M.S.; Spectrophotometric methods for simultaneous determination of nitazoxanide and ofloxacin in combined bulk and pharmaceutical formulations; International Journal of PharmTech Research, (2011); 3: 118– 123. Sagar, V., Nizami, S.A., Gurumurthy, M., Ramesh, R.; Colorimetric estimation of nitazoxanide and ofloxacin in pharmaceutical dosage form; International Journal of Pharmaceutical Research and Development, (2011); 3: 199–206. Madhuri, D.G., Sakarkar, D.M.; Simultaneous spectrophotometric estimation of nitazoxanide and ofloxacin in tablets; Indian Journal of Pharmaceutical Sciences, (2011); 73: 70– 74. Sharma, S., Sharma, M.C.; Development of visible spectrophotometric methods for the estimation of nitazoxanide in bulk and pharmaceutical formulation using ferric chloride; American-Eurasian Journal of Scientific Research, (2011); 6: 155– 160. EL-Desoky, H.S., Ghoneim, M.M., Abdel-Galeil, M.M.; Stripping voltammetric methods for determination of the antiparasitic drug nitazoxanide in bulk form, pharmaceutical formulation and human serum; Journal of the Brazilian Chemical Society, (2010); 21: 669–679. International Conference of Harmonization of technical requirements for registration of pharmaceuticals for human use (ICH), www.nihs.go.jp (accessed November 14, 2013).
Validated Stability Indicating RP-HPLC Method 11
Article
Validated Stability Indicating RP-HPLC for Quantitation of Nitazoxanide in Presence of Its Alkaline Degradation Products and Their Characterization by HPLC-Tandem Mass Spectrometry Maha Hegazy1*, Amira Kessiba2, Ahmed Emad El Gindy2 and Mohamed Abdelkawy1 1
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, 11562 Cairo, Egypt and 2Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, KM 28 Cairo – Ismailia Road (Ahmed Orabi District), Cairo, Egypt
*Author to whom correspondence should be addressed. Email: [email protected] Received 5 February 2013; revised 14 October 2013
A simple and sensitive stability indicating HPLC method was developed and validated for quantitative determination of Nitazoxanide (NTZ), a new antiprotozoal drug, in presence of degradation products generated under forced alkaline hydrolysis. Chromatographic separation was achieved on Inertsil C8-3 column (150 3 4.6 mm i.d.) using a mobile phase composed of acetonitrile: 50 mM ammonium acetate buffer (50:50, v/v, pH 5.0 adjusted with acetic acid) at a flow rate of 1 mL/min. Quantification was achieved with UV detection at 298 nm based on relative peak area. The method was linear over the concentration range of 0.8–50 mg/mL (r 5 0.9999) with a limit of detection and quantification 0.0410 and 0.1242 mg/mL, respectively. The developed method has the requisite accuracy, selectivity, sensitivity and precision to assay NTZ in presence of its degradation products either in bulk powder or in pharmaceutical formulations. The degradation products were then identified by HPLC-MS/MS analysis using an electrospray ionization source and an ion trap analyzer.
lower sensitivity of these methods as they determined NTZ at higher concentration levels than the present work. The present work focuses on detailed study of the alkaline degradation of NTZ due to its high instability in alkaline medium and our aim was to develop a simple and sensitive stability indicating the RP-HPLC method for the quantitative determination of NTZ in presence of its alkaline degradation products and the method was validated according to ICH guidelines (47). The degradation products were characterized by liquid chromatographytandem mass spectrometry analysis using an electrospray ionization (ESI) source and an ion trap analyzer. Experimental Instruments HPLC instrumentation and conditions Samples were loaded into Rheodyne 7725i injection valve, equipped with a 20-mL sample loop (Rheodyne, Berkeley, CA,
Introduction Nitazoxanide (NTZ), as shown in Figure 1A, is a nitrothiazole benzamide compound that has a wide range of antimicrobial activity against parasites, bacterial and viral pathogens. The broad spectrum of in vivo activity is related to its desacetyl derivative, tizoxanide (Figure 1B), and includes intracellular and extracellular protozoa, helminthes, aerobic and anaerobic bacteria and virus (1 –8). It is chemically designated as N-(5-nitro-2-thiazolyl) salicyl amide acetate (9). It is prescribed for treatment of diseases caused by Giardia intestinalis and Cryptosporidium in immune-compromised patients, including those with AIDS or HIV infection. The drug represents a significant advance in the treatment of intestinal parasitical infections worldwide (10, 11). NTZ is not an official drug. Several chromatographic (12 –31), spectrophotometric (32 –45) and electrochemical (46) analytical methods have been reported for the determination of NTZ in bulk powder, pharmaceutical formulations alone or in combination with other drugs and/or in biological fluids. Several stability indicating HPLC methods (13– 15, 22) have been reported for the determination of NTZ in the presence of its degradation products. These methods showed that NTZ undergoes slight degradation when exposed to acid, oxidative and photodegradation, but it is highly unstable in alkaline medium and is stable to heat. None of these methods provided identification of the degradation products formed as well as the
Figure 1. The chemical structure of Nitazoxanide (NTZ) (A). The chemical structure of tizoxanide (B).
# The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
USA). HPLC separation and quantitation were made on Inertsil C8-3 column (150 4.6 mm i.d., 5 mm particle size) with a mobile phase consisting of acetonitrile: 50 mM ammonium acetate buffer (50:50, v/v), pH was adjusted to 5.0 using acetic
acid. The mobile phase was filtered using 0.45 mm membrane filters (Millipore, Milford, MA, USA) and degassed by ultrasonic vibrations for 15 min prior to use. An isocratic pump was used to deliver the mobile phase at a flow rate of 1 mL/min (Agilent 1100
Figure 2. Typical HPLC chromatograms of (A) pure NTZ 10 mg/mL (tR ¼ 5.295), (B) alkaline degradation products formed after 15 min reflux with 0.1 N NaOH (NTZ DegI) equivalent to 10 mg/mL (tR ¼ 1.882, 2.191, 2.635), (C) alkaline degradation products formed after 1 h reflux with 0.1 N NaOH (NTZ DegII) equivalent to 10 mg/mL (tR ¼ 1.882, 2.639).
2 Hegazy et al.
Series Iso pump G1310A, Agilent Technologies, Santa Clara, CA, USA). The samples were filtered using 0.45 mm membrane filters. The UV–Vis detector (Agilent 1100 Series VWD G1314A) was set at 298 nm. Data acquisition were performed on Agilent LC ChemStation software. All determinations were performed at ambient temperature (258C). Liquid chromatography–tandem mass spectrometry instrumentation and conditions Liquid chromatography-tandem mass spectrometry (LC–MS/MS) composed of Applied Biosystem 3200 Q trap with LC consisting of a solvent delivery system and an autosampler (Agilent 1200). The mobile phase consisted of acetonitrile:10 mM ammonium acetate buffer (pH ¼ 4.5) in the ratio of 50:50 at a flow rate 1 mL/min. The mobile phase containing the analytes was flowing directly into a positive and negative electrospray ionization probe (+ESI)
of a tandem triple quadruple mass spectrometer (Q trap). The instrument permits various scanning modes such as MS, MS/MS. Tuning parameters for MS scan for NTZ were optimized in negative mode by direct infusion of NTZ solution at a concentration of 1 mg/mL. The capillary voltage was adjusted at 4 kV, collision energy at 13 eV and collision gas pressure at 3 mbar. All data acquisition were controlled by Analyst 1.5 software.
Materials Pure standard NTZ standard material was supplied by Sigma Pharmaceutical Industries, 1st zone, Mubarak industrial zone, Quwasnah, El Monofeya-Egypt, it was certified to contain 99.5% (w/w) according to the manufacturer’s method.
Figure 3. HPLC chromatograms of (A) resolved mixture of 10 mg/mL of NTZ (tR ¼ 5.302), and 10 mg/mL of its alkaline degradate (NTZ DegI) (tR ¼ 1.879, 2.188, 2.633), (B) resolved mixture of 10 mg/mL of NTZ (tR ¼ 5.299), and 10 mg/mL of its alkaline degradate (NTZ DegII) (tR ¼ 1.879, 2.634) mobile phase is acetonitrile: 50 mM ammonium acetate buffer (50:50, v/v, pH adjusted to 5.0 using acetic acid).
Validated Stability Indicating RP-HPLC Method 3
Pharmaceutical formulations (i) Nanazoxid w tablets (Batch No. 10206) are labeled to contain 500 mg of NTZ per tablet, produced by Medizen Pharmaceuticals, Borg El arab-Alex.-Egypt for Utopia Pharmaceuticals. (ii) Nitazode w suspension (Batch No. 02785) is labeled to contain 100 mg of NTZ per 5 mL suspension, manufactured by Sigma Pharmaceutical Industries for Al Andalous Medical Co., Cairo, Egypt. Chemicals and reagents All chemicals and reagents used throughout this work were of analytical grade and solvents were of HPLC grade: (i) Sodium hydroxide (Adwic, Egypt) (ii) Ammonium acetate (Adwic, Egypt) (iii) Acetic acid (Adwic, Egypt). (iv) Hydrochloric acid (Sigma-Aldrich, Germany). (v) Acetonitrile (Sigma-Aldrich, Germany). Solutions NTZ stock standard solution NTZ standard solution (100 mg/mL) was prepared by accurately weighing 10 mg of NTZ into 100 mL volumetric flask, dissolve in and complete to volume with acetonitrile. The solution was protected from light by wrapping the flask with aluminum foil. Alkaline degradation products stock solution An accurately weighed amount of pure NTZ (10 mg) was transferred into two separate conical flasks, the first one was refluxed with 20 mL of 0.1 N NaOH for 15 min (NTZ DegI) and the second one was refluxed with 20 mL of 0.1 N NaOH for 1 h (NTZ DegII). The solutions were then neutralized by adjusting the pH using 0.1 N HCl and transferred quantitatively to 100 mL volumetric flasks. The volumes were then completed using acetonitrile to produce concentration equivalent to 100 mg/mL of each. The degradation process was followed every 15 min and complete degradation was confirmed by HPLC using 50 mM ammonium acetate buffer: acetonitrile (50:50, v/v, and the pH was adjusted to 5.0 using acetic acid). Laboratory prepared mixtures Aliquots (1.8–0.2 mL) of NTZ standard solution (100 mg/mL) equivalent to 18 –2 mg/mL were accurately transferred into two series of 10 mL volumetric flasks, to the first series aliquots
Table II Accuracy Results of the Proposed HPLC Method for the Determination of NTZ Pure Samples NTZ (mg/mL)
Recovery %
Taken
Found
2 5 8 15 18 25 35 45 Mean RSD%
2.01 5.02 7.99 14.91 17.96 25.11 34.96 45.15
100.5 100.4 99.9 99.4 99.8 100.4 99.9 100.3 100.1 0.397
Table III Determination of NTZ in Presence of Its Alkaline Degradation Products (NTZ DegI and NTZ DegII) in Laboratory Prepared Mixtures by the Proposed HPLC Method Mixture no.
NTZ DegI %
NTZ (mg/mL) Taken
Found
Recovery %
1 2 3
10 50 90
18 10 2
17.72 10.05 2.01
98.4 100.5 100.5
Mean RSD%
99.8 1.192
NTZ DegII % 10 50 90
NTZ (mg/mL) Taken
Found
Recovery %
18 10 2
17.79 9.97 2.03
98.8 99.7 101.5
Mean RSD%
100.0 1.362
Table IV Results Obtained by Applying the Proposed HPLC Method for the Determination of NTZ in Nanazoxidw Tablets and Nitazodew Suspension and Results Obtained by Applying the Standard Addition Technique Product
Found*% + RSD%
w
Nanazoxid tablets 500 mg NTZ/tablet B.N.10206
99.1 + 0.497
Nitazodew suspension 100 mg NTZ/5 mL B.N.02785
98.9 + 1.151
Standard addition technique Added (mg/mL)
Recovery%
18 20 22 Mean 100.6 RSD% 0.667 18 ‘ 22 Mean 99.1 RSD% 0.546
100.0 100.4 101.3
98.5 99.5 99.4
*Average of four determinations.
Table I Validation Parameters for the Determination of Pure NTZ Samples by the Proposed HPLC Method Parameter
NTZ
Range Slope Intercept SE of the slope SE of the intercept Correlation coefficient (r) LOD LOQ Precision Repeatability (%) Intermediate precision (%)
0.8 –50 mg/mL 0.1009 0.0079 0.0003 0.0102 1 0.0410 0.1242
4 Hegazy et al.
0.713 0.787
Table V Statistical Comparison of the Results Obtained by the Proposed HPLC Method and the Reported HPLC Method(B) for the Analysis of NTZ in Pure Form Parameter
NTZ
Reported HPLC method
Mean SD n Variance Student’s t F
100.1 0.397 8 0.158 0.548 (2.447)* 2.273 (4.120)*
100.2 0.598 5 0.358
*The values between parentheses are the tabulated values for t and F at p ¼ 0.05.
(0.2 –1.8 mL) of NTZ DegI stock solution (100 mg/mL) were accurately added and to the second one, similar aliquots of NTZ DegII stock solution (100 mg/mL) equivalent to 2 –18 mg/mL were accurately added. The volumes were then completed with the mobile phase to prepare mixtures containing from 10 to 90% of NTZ DegI and NTZ DegII. Solutions for LC –ESI –MS/MS analysis An aliquot of each of NTZ standard solution and NTZ degradation products (NTZ DegI, NTZ DegII) stock solutions was diluted with mobile phase to give a final concentration of 1 mg/ mL. The prepared samples were injected into LC –MS using positive and negativeESI. The MS and MS/MS scans were selected for the identity studies.
Procedure Calibration of standard solutions Different aliquots (0.08–5 mL) of NTZ standard solution (100 mg/mL), equivalent to 0.8– 50 mg/mL, were transferred
into a series of 10 mL volumetric flasks and the volumes were adjusted with the mobile phase. An aliquot of 20 mL of each solution was injected into the chromatographic system and processed according to the previously described conditions.
Application to pharmaceutical formulations Nanazoxidw tablets. Six Nanazoxid tablets (labeled to contain 500 mg NTZ) were accurately weighed and finely powdered. An accurate weight of the powdered tablets equivalent to 10 mg NTZ was transferred into a 100-mL volumetric flask, extracted with 50 mL acetonitrile in an ultrasonic bath for 10 min and diluted to volume with the same solvent. The solution (100 mg/mL) was shaken again for 10 min in the ultrasonic bath and filtered. Different aliquots (0.4, 0.6 and 1 mL) were transferred into a series of 10 mL volumetric flasks and the volumes were completed with the mobile phase to produce solutions of concentrations equivalent to 4, 6 and 10 mg/mL of NTZ, respectively. The procedure was completed as described under calibration and the concentrations of NTZ were calculated from the corresponding regression equation.
Figure 4. (A) MS spectrum of NTZ solution corresponding to the peak at tR ¼ 5.29 in Figure 2A), (B) MS/MS product ion spectrum of m/z 305.9 corresponding to NTZ.
Validated Stability Indicating RP-HPLC Method 5
Nitazodew suspension. The whole bottle was emptied and the powder was accurately weighed. An accurately weighed amount of powder equivalent to 10 mg NTZ was transferred into a 100-mL volumetric flask, then the procedure was completed as described above.
Results and discussion The literature survey revealed that NTZ is liable to acid, alkaline, oxidative and photodegradation (12– 15, 22, 24). In this work, we are focusing on the alkaline degradation of NTZ as it shows a high degree of instability in alkaline medium.
HPLC analysis Nitazoxanide was subjected to hydrolysis under alkaline-stress conditions by refluxing with 0.1 N NaOH and degradation was followed every 15 min by HPLC [C18 column, mobile phase: 50 mM ammonium acetate buffer– acetonitrile (50:50, v/v), pH adjusted to 5.0 using acetic acid and detection at 298 nm]. It was found that complete degradation occurred after 15 min (NTZ
DegI) with the formation of three degradation products. By following the degradation procedure, it was found that the peak area corresponding to one of the formed degradates decreased gradually with the increase in the area of one of the other two peaks. After 1 h (NTZ DegII), two final degradation products were obtained without further change. Representative chromatograms showing the degradation process are shown in Figure 2. In order to optimize the proposed HPLC method, several trials were carried out to obtain good and optimum separation of NTZ from its degradation products. Initially, methanol and acetate buffer in different ratios as (60:40 and 65:35, v/v) were tried, but this showed peak broadening. Acetonitrile was tried with methanol and acetate buffer in the ratio (30:30:40, v/v/v), but this resulted in decreased peak symmetry, so methanol was totally replaced by acetonitrile. The effect of changing the ratio of the organic modifier on the retention times was investigated and finally, it was found that acetonitrile:acetate buffer in the ratio (50:50, v/v, pH adjusted to 5.0 using acetic acid) with a flow rate of 1 mL/min was most suitable to get resolved and sharp peaks. The optimum wavelength for detection and quantification was 298 nm, at which good detector response was obtained with symmetrical peaks.
Figure 5. MS spectra of (A) NTZ alkaline degradate (NTZ DegI) corresponding to the peaks at tR ¼ 1.882 (m/z 136.9) and tR ¼ 2.191 (m/z 178.9) in Figure 2B in negative ion mode, (B) NTZ DegI corresponding to the peak at tR ¼ 2.635 in Figure 2B in positive ion mode. 6 Hegazy et al.
Figure 6. MS/MS product ion spectra of (A) m/z 178.9 in negative mode, (B) m/z 136.9 in negative mode, (C) m/z 146.1 in positive mode.
Validated Stability Indicating RP-HPLC Method 7
Upon applying the previously described HPLC optimum experimental conditions, good and efficient separation was observed between NTZ and its alkaline degradation products (NTZ DegI and NTZ DegII), Figure 3. Linear relationship was obtained for NTZ between the relative peak areas and the corresponding concentrations, Supplementary data, Figure S1. The regression equation was computed and found to be: A ¼ 0:1008C þ 0:0067; r ¼ 0:9999 where A is the relative peak area, C is NTZ concentration in mg/mL and r is the correlation coefficient.
System suitability was checked by calculating different parameters such as capacity factor, tailing factor, column efficiency (N), selectivity and resolution factors, where the system was found to be suitable relative to the reference values, Supplementary data, Table S1. Validation (47) of the proposed method was constructed by determining the linearity, range, accuracy and precision (Table I). The proposed HPLC method was successfully applied for the determination of NTZ in pure powdered form with mean percentage recoveries of 100.1 + 0.397% (Table II).
Figure 7. MS spectra of (A) NTZ alkaline degradate (NTZ DegII) corresponding to the peak at tR ¼ 1.882 in Figure 2C in negative mode, (B): NTZ DegII corresponding to the peak at tR ¼ 2.639 in Figure 2C in positive mode.
8 Hegazy et al.
The specificity of the proposed method was proved by the analysis of laboratory prepared mixtures containing different ratios of NTZ-NTZ DegI and NTZ-NTZ DegII, satisfactory results were obtained for NTZ in the presence of up to 90% of both NTZ DegI and NTZ DegII (Table III). The proposed HPLC method has been successfully applied for the determination of NTZ in Nanazoxidw tablets and Nitazodew suspension. The validity of the method was further assessed by applying the standard addition technique (Table IV). The results obtained for the analysis of NTZ in its pure form by the proposed HPLC method were statistically compared with those obtained by applying a reported HPLC method (14). The calculated t and F values were less than the tabulated ones at 95% confidence level, which reveals that there is no significant difference between the two methods with respect to accuracy and precision (Table V).
Identification of degradation products by LC– ESI–MS/MS analysis The MS scan of a reference solution of NTZ in negative ion mode showed a strong signal at m/z 305.9 [M2H]2 which refers to the deprotonated molecular (parent) ion of the drug (Figure 4A). The MS/MS scan of NTZ showed daughter ions at m/z (305.9, 263.9 and 185.9) (Figure 4B), the signal formed at m/z 263.9 [M2H]2 was assigned to tizoxanide (Figure 1B), which is the active metabolite of NTZ formed through de-acetylation of the parent ion. Scanning of NTZ DegI solution in negative ion mode showed two strong signals at m/z 178.9 [M2H]2 and 136.9 [M2H]2 (Figure 5A), which were assigned to acetyl salicylic acid and salicylic acid, respectively. By scanning NTZ DegI solution in positive ion mode, a signal appeared at m/z 146.1 [M2H]þ (Figure 5B), which corresponds to 5-nitro-1,3-thiazol-2-amine resulting from the hydrolysis of the amide linkage of NTZ. The MS/MS scan of
Figure 8. Schematic diagram showing the suggested NTZ degradation products.
Validated Stability Indicating RP-HPLC Method 9
the product ion at m/z 178.9 [M2H]2 corresponding to acetylsalicylic acid showed a major fragment in negative mode at m/z 135.9 [M2H]2 resulting from the loss of the acetyl group (Figure 6A). The ion at m/z 136.9 [M2H]2 corresponding to salicylic acid shows a major fragment at m/z 93 [M2H]2 in the MS/ MS scan in negative ion mode (Figure 6B). By fragmentation of the ion at m/z 146.1 [M2H]þ in positive mode, it showed a major fragment at m/z 105 [M2H]þ in the MS/MS spectrum (Figure 6C). Scanning NTZ DegII solution in negative ion mode showed a strong signal at m/z 136.9 [M2H]2 (Figure 7A), which was assigned to salicylic acid as described above, and in positive ion mode, it showed a strong signal at m/z 146.1 [M2H]þ (Figure 7B), corresponding to 5-nitro-1,3-thiazol-2-amine. Tuning parameters for MS and MS/MS scans of NTZ and its alkaline degradation products are listed in Supplementary data, Table S2. The degradation pathway of NTZ and the structures of the degradation products formed are shown in Figure 8. It was found that the main degradation route of NTZ is via hydrolysis of the amide linkage and the ester linkage. Upon refluxing of NTZ with 0.1 N NaOH for 15 min (NTZ DegI), complete degradation occurred through hydrolysis of the amide linkage and partial hydrolysis of the ester linkage giving three degradation products which are acetyl salicylic acid, salicylic acid and 5-nitro-1,3thiazol-2-amine. By continuing the degradation procedure, gradual de-acetylation of the formed acetyl salicylic acid occurred giving salicylic acid, which explains the gradual decrease in the peak area of one of the degradation products with the increase in the area of the other one in HPLC. After 1 h, only two degradation products were detected, which are salicylic acid and 5-nitro-1,3-thiazol-2-amine. So NTZ must be properly stored to avoid the presence of salicylic acid resulting from the degradation process, which exposes the patient to toxicity.
Conclusion The suggested method is simple, accurate, selective and sensitive. Application of the proposed method to the analysis of NTZ in laboratory prepared mixtures and pharmaceutical formulations showed that neither the degradation products nor the excipients interfere with the determination, indicating that the proposed method could be applied as stability indicating method for the determination of NTZ in presence of its degradation products either in bulk powder or in pharmaceutical formulations. Also identification of the degradation products was successfully achieved by LC –ESI –MS/MS analysis.
Supplementary data Supplementary data are available at Journal of Chromatographic Science online. References 1. Dubreuil, L., Houcke, I., Mouton, Y., Rossignol, J.F.; In vitro evaluation of activities of nitazoxanide and tizoxanide against anaerobes and aerobic organisms; Journal of Antimicrobial Agents and Chemotherapy, (1996); 40: 2266–2270.
10 Hegazy et al.
2. Cabello, R.R., Guerrero, L.R., Garcia, M.R.M., Cruz, A.G.; Nitazoxanide for the treatment of intestinal protozoan and helminthic infections in Mexico; Journal of Transactions of the Royal Society of Tropical Medicine and Hygiene, (1997); 91: 701–703. 3. Megraud, F., Occhialini, A., Rossignol, J.F.; Nitazoxanide a potential drug for eradication of Helicobacter pylori with no cross-resistance to metronidazole; Journal of Antimicrobial Agents and Chemotherapy, (1998); 42: 2836–2840. 4. Rossignol, J.F., Ayoub, A., Ayers, M.S.; Treatment of diarrhea caused by Cryptosporidium parvum: a prospective randomized, double-blind, placebo-controlled study of nitazoxanide; Journal of Infectious Diseases, (2001); 184: 103–106. 5. Rossignol, J.F., Ayoub, A., Ayers, M.S.; Treatment of diarrhea caused by Giardia intestinalis and Entamoeba histolytica or Entamoeba dispar: a randomized, double-blind, placebo-controlled study of nitazoxanide; Journal of Infectious Diseases, (2001); 184: 381–384. 6. Rossignol, J.F., Abou Zekry, M., Hussein, A., Santoro, M.G.; Effect of Nitazoxanide in treating severe Rotavirus diarrhea: a randomized, double-blind, placebo-controlled trial; Lancet, (2006); 368: 124– 129. 7. Esposito, M., Mu¨ller, N., Hemphill, A.; Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum; International Journal of Parasitology, (2007); 37: 183– 190. 8. Korba, B.E., Montero, A.B., Farrar, K., Gaye, K., Mukerjee, S., Ayers, M.S., et al.; Nitazoxanide, tizoxanide and other thiazolides are potent inhibitors of hepatitis B virus and hepatitis C virus replication; Journal of Antiviral Research, (2008); 77: 56– 63. 9. Budavaried, S. The Merck Index, an Encyclopedia of Chemicals, Drugs and Biologicals. (14th ed.). Merck & Co., Inc., Whitehouse Station, NJ, USA, (2006). 10. Sweetman, S.C. Martindale: The Complete Drug Reference. (33rd ed.) The Pharmaceutical Press, London, (2002), pp. 598– 599. 11. Stockis, A., Deroubaix, X., Lins, R., Jeanbaptiste, B., Calderon, P., Rossignol, J.F.; Pharmacokinetics of nitazoxanide after single oral dose administration in 6 healthy volunteers; International Journal of Clinical Pharmacology and Therapeutics, (1996); 34: 349–351. 12. Gopu, C.L., Thomas, S., Paradkar, A.R., Mahadik, K.R.; A validated stability indicating HPTLC method for determination of nitazoxanide; Journal of Scientific & Industrial Research, (2007); 66: 141–145. 13. Jadhav, A.S., Pathare, D.B., Shingare, M.S.; A validated stability indicating RP-LC method for nitazoxanide, a new antiparasitic compound; Chromatographia, (2007); 66: 595– 600. 14. Malesuik, M.D., Cardoso, S.G., Steppe, M.; Development of a validated stability indicating LC method for nitazoxanide in pharmaceutical formulations; Chromatographia, (2008); 67: 131–136. 15. Rane, V.P., Sangshetti, J.N., Patil, K.R., Yeole, R.D., Shinde, D.B.; Stability indicating LC determination of nitazoxanide in bulk drug and in pharmaceutical dosage form; Chromatographia, (2008); 67: 455– 459. 16. Sakamoto, T., Hiyama, Y.; Rapid determination of nitazoxanide in tablets using reversed-phase ultra-performance liquid chromatography and high performance liquid chromatography; Die Pharmazie, (2008); 63: 503–507. 17. Kalta, R.R., Sharma, R., Chaturvedi, S.C.; Simultaneous RP-HPLC determination of nitazoxanide and ofloxacin in combined tablet dosage form; Indian Journal of Pharmaceutical Sciences, (2008); 70: 491–494. 18. Zhao, Z., Zhang, L., Xue, F., Wang, X., Zheng, W., Zhang, T., et al.; Liquid chromatography-tandem mass spectrometry analysis of nitazoxanide and its major metabolites in goat; Journal of Chromatography B, (2008); 875: 427–436. 19. Jadhav, V.Y., Gandhi, S.V., Dhavale, N.D., Sabnis, S.S.; RP-HPLC determination of nitazoxanide in bulk and different tablet formulations; Eurasian Journal of Analytical Chemistry, (2008); 3: 404–409. 20. Kumar, R.S., Nallasivan, P.K., Saravanakumar, S., Kandasamy, C.S., Venkatnarayanan, R.; Simultaneous RP-HPLC estimation of nitazoxanide and ofloxacin in tablet dosage forms; Asian Journal Research Chemistry, (2009); 2: 43 –45. 21. Ruiz-Olmedo, M.I., Gallegos-Perez, J.L., Calderon-Gonzalez, K.G., Franco-Perez, J., Jung-Cook, H.; Sensitive high performance liquid
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
chromatographic assay for nitazoxanide metabolite in plasma; Die Pharmazie, (2009); 64: 419–422. Malesuik, M.D., Goncalves, H.M.L., Paim, C.S., Schapoval, E.E.S., Steppe, M.; LC: analysis of photodegradation kinetics of nitazoxanide in pharmaceutical formulations; Journal of Chromatographic Science, (2009); 47: 745– 748. Vipul, P.R., Devanand, B.S.; Simultaneous high performance liquid chromatographic determination of nitazoxanide and ofloxacin in tablet formulation; Eurasian Journal of Analytical Chemistry, (2009); 4: 304–310. Malesuik, M.D., Goncalves, H.M.L., Paim, C.S., Jablonski, A., Schapoval, E.E.S., Steppe, M.; Stability indicating CZE method and stress degradation studies of nitazoxanide; Journal of Liquid Chromatography and Related Technologies, (2010); 33: 375–389. Valarmathy, J., Samueljoshua, L., Rathinavel, G., Senthilkumar, K.L.; Method development and validation for assay of nitazoxanide in tablet using RP-HPLC; International Journal of ChemTech Research, (2010); 2: 1263–1266. Premanand, D.C., Senthilkumar, K.L., Senthilkumar, B., Saravanakumar, M., Thirumurthy, R.; A validated RP-HPLC method for simultaneous estimation of nitazoxanide and ofloxacin in pharmaceutical formulation; Der Chemica Sinica, (2010); 1: 1– 5. Singh, H.P., Agal, S.K., Sharma, C.S., Ranawat, M.S., Ankalgi, A.D.; RP-HPLC methods for estimation of nitazoxanide single and simultaneous estimation of nitazoxanide with ofloxacin in pharmaceutical dosage forms; Journal of Chemical and Pharmaceutical Research, (2010); 2: 984–992. Sharma, S., Bhandari, A., Choudhary, V.R., Rajpurohit, H., Khandelwal, P.; RP-HPLC method for simultaneous estimation of nitazoxanide and ofloxacin in tablets; Indian Journal of Pharmaceutical Sciences, (2011); 73: 84 –88. Bhale, S., Patankar, R.D.; RP-HPLC development and validation of nitazoxanide in tablet dosage form; International Journal of Pharmacy and Pharmaceutical Sciences, (2011); 3: 71– 73. Sharma, M.C., Sharma, S.; Development and validation of a dissolution method with isocratic high performance liquid chromatographic determination of nitazoxanide and ofloxacin in pharmaceutical dosage form; American-Eurasian Journal of Scientific Research, (2011); 6: 182–187. Mehta, D., Seth, A.K., Kumar, S., Yadav, Y.C.; HPLC method for simultaneous estimation of ofloxacin and nitazoxanide in its tablet dosage form; Pharma Science Monitor, (2011); 2: 1 –12. Kapse, G.K., Prabhakar, G., Raju, S.A.; Spectrophotometric methods for the estimation of nitazazoxanide in pharmaceutical formulations; Indian Journal of Pharmaceutical Sciences, (2006); 68: 403– 406. Lakshminarayana, K.V., Manohara, Y.N., Gurupadayya, B.M.; Development and validation of spectrophotometric methods for the estimation of nitazoxanide in tablet dosage forms; Indian Journal of Pharmaceutical Sciences, (2007); 69: 147–149. Senthilraja, M.; Simultaneous UV spectrophotometric method for the estimation of nitazoxanide and ofloxacin in combined dosage form;
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
Research Journal of Pharmacy and Technology, (2008); 1: 469– 471. Vanitha, J., Kumar, A., Ganesh, M., Vetrichelvan, T.; Estimation of nitazoxanide by UV spectrophotometric method; Journal of Global Pharma Technology, (2009); 1: 51 –53. EL-Desoky, H.S., Ghoneim, M.M., Abdel-Galeil, M.M.; A direct spectrophotometric method for determination of antiparasitic drug nitazoxanide in a bulk form and pharmaceutical formulation; Chemia Analityczna, (2009); 54: 1065–1077. Sherje, A.P., Chokshi, J., Satam, C.A., Chaudhary, D., Vanshiv, S.D.; Spectrophotometric determination of nitazoxanide and ofloxacin in combined dosage form; Journal of Pharmacy Research, (2010); 3: 72– 74. Malesuik, M.D., Paim, C.S., Schapoval, E.E.S., Steppe, M.; Development of a simple, rapid and validated spectrophotometric method for nitazoxanide in pharmaceutical formulations and comparison with HPLC; Quimica Nova, (2010); 33: 739–742. Bindaiya, S., Bankey, S., Jain, D.; Simultaneous determination of nitazoxanide and ofloxacin in tablet by ultraviolet spectrophotometry (dual wavelength method); International Journal of ChemTech Research, (2010); 2: 11– 15. Sherje, A.P., Anand, R., Sriram, W., Vanshiv, S.D.; Mixed hydrotropy in spectrophotometric analysis of nitazoxanide; International Journal of ChemTech Research, (2010); 2: 1966–1969. Adhikari, L., Sahu, S., Jagdev, S.; Development and validation of spectrophotometric and colorimetric method for the determination of nitazoxanide in its bulk and pharmaceutical dosage form; International Journal of ChemTech Research, (2011); 3: 131– 135. Singh, H.P., Sharma, C.S., Ankalgi, A.D., Agal, S.K., Ranawat, M.S.; Spectrophotometric methods for simultaneous determination of nitazoxanide and ofloxacin in combined bulk and pharmaceutical formulations; International Journal of PharmTech Research, (2011); 3: 118– 123. Sagar, V., Nizami, S.A., Gurumurthy, M., Ramesh, R.; Colorimetric estimation of nitazoxanide and ofloxacin in pharmaceutical dosage form; International Journal of Pharmaceutical Research and Development, (2011); 3: 199–206. Madhuri, D.G., Sakarkar, D.M.; Simultaneous spectrophotometric estimation of nitazoxanide and ofloxacin in tablets; Indian Journal of Pharmaceutical Sciences, (2011); 73: 70– 74. Sharma, S., Sharma, M.C.; Development of visible spectrophotometric methods for the estimation of nitazoxanide in bulk and pharmaceutical formulation using ferric chloride; American-Eurasian Journal of Scientific Research, (2011); 6: 155– 160. EL-Desoky, H.S., Ghoneim, M.M., Abdel-Galeil, M.M.; Stripping voltammetric methods for determination of the antiparasitic drug nitazoxanide in bulk form, pharmaceutical formulation and human serum; Journal of the Brazilian Chemical Society, (2010); 21: 669–679. International Conference of Harmonization of technical requirements for registration of pharmaceuticals for human use (ICH), www.nihs.go.jp (accessed November 14, 2013).
Validated Stability Indicating RP-HPLC Method 11
