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Background: Aza sugars are organi c sugars having nitrogen containing polyhydroxyl sugar molecules. These molecules are active pharmaceutical ingredients; these are not well separated and eluted early in the HPLC and UPLC columns due to high polar nature. Aza sugars are having high conductivity hence the ion chromatography validated method has been established for the castanospermin and celgosivir along with its degradation studies (impurities). Methods: An ion chromatography with conductivity detector-cation column was used to determine the assay of castanospermin and celgosivir as in the form of bulk active pharmaceutical ingredients. The degradation impurities were identified and characterized by using the UPLC-TOF and the LCMS/MS techniques. Results: An ion chromatography method was developed and determined the assay for castanospermin and celgosivir as in the form of bulk active pharmaceutical ingredients with the specificity of the miglitol and 1- deoxynojirimycin. Validation was performed for assay of the castanospermin and celgosivir. The method precision %RSD results at 0.25 mg/mL concentration of castanospermin and celgosivir were 1.1 and 0.7 respectively. The linearity was performed from 25 to 200% (w.r.t 0.25 mg/mL); the results were 1.000 and 0.999 coefficient for the castanospermin and celgosivir respectively. The recovery studies, robustness, ruggedness, and solution stability results were within the acceptance limits of the ICHQ2 (R1) guidelines. The stress study for the castanospermin and celgosivir active pharmaceutical ingredients was performed by using 0.5N HCl solution, 0.5N NaOH solution, 3.0% H202 solution, UV-visible and the thermal conditions. The castanospermin was degraded as 20.8% of n- oxide impurity, and celgosivir was degraded as 10.0% n-oxide impurity under 3.0% H202 solution. In base degradation, the celgosivir was back converted completely to castanospermin. These n-oxide impurities were identified and characterized by using UPLC-TOF and LCMS/MS techniques after collection from the ion chromatography. Castanospermin and celgosivir are stable in remaining stress conditions. Conclusions: From the present study, it was found that robust analytical ion chromatography technique is used for the determination of assay in Aza sugar, especially assay for the castanospermin and celgosivir with minimum usageoftestsample0.25mg/mL andusedgreen chemistry solvents. The study also explains that the unique degradation of castanospermin and celgosivir under oxidative and base hydrolysis, Oxidative degradation impurities were identified and characterized as n-oxides of its respective castanospermin and celgosivir active pharmaceutical ingredients by using HRMS and LC-MS/MS. * Correspondence: firstname.lastname@example.org Analytical Research and Development, Dr. Reddy’s Laboratories, Hyderabad, Telangana 500049, India Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 2 of 16 Fig. 3 Blend chromatograms of selected Aza sugars with Metrosep Fig. 1 UPLC method chromatogram of Aza sugars C4 250/4.0 column Background development by Migenix for the treatment of HCV infec- 1-Deoxynojirimycin, miglitol, castanospermin, and celgosi- tion; it is an oral prodrug of the natural product. Castanos- vir are some of the selected Aza sugars, which are now permin inhibits alpha-glucosidase, an enzyme that used for the treatment of diabetes, dengue, and hepatitis C plays a critical role in viral maturation by initiating virus (HCV) diseases respectively. Celgosivir is in the processing of the N-linked oligosaccharides of viral envelope glycoproteins. Celgosivir is well Fig. 2 Development of Aza sugars (1-deoxynojirimycin, miglitol, Fig. 4 Blend chromatograms of selected Aza sugars (1- castanospermin, and celgosivir) with universal cation column in deoxynojirimycin, miglitol, castanospermin, and celgosivir) with ion chromatography Metrosep C4 150/4.0 column Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 3 of 16 absorbed in vitro and in vivo and rapidly gets con- inhibitor of alpha-glucosidase (Belley et al. 2013), a verted to castanospermin. Celgosivir has a novel host enzyme required for viral assembly, release, and mechanism of action and demonstrates broad antiviral infectivity. Castanospermin is an indolizidine alkaloid activity in vitro. Celgosivir is not efficient as a mono- first isolated from the seeds of Castanospermum aus- therapy for the treatment of HCV but has a synergis- trale. It is a potent inhibitor of some glucosidase en- tic effect in combination with pegylated interferon zymes and has antiviral activity in vitro and in mouse alfa-2b plus ribavirin, both in vitro and in phase II models. 1-Deoxynoijirimycin is also called as duvoglu- clinical trials that last up to 1 year in patients with stat or moranolin. It is an inhibitor; it is mostly chronic HCV infection. Celgosivir is a 6-0-butanoyl present in the mulberry leaves and also present by ester derivative of castanospermin, a compound de- brewing little quantities from the (herbal tea) of the rived from the Australian chestnut with activity mulberry leaves. Miglitol is an oral anti-diabetic drug; against hepatitis C virus. Celgosivir rapidly converts it is acting as an inhibitor, the ability to break down to castanospermin in the body, where it is a potent complex carbohydrates into glucose. It is used in Fig. 5 HR-MS spectrum of celgosivir and its n-oxide impurity Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 4 of 16 diabetes mellitus type 2 for establishing greater gly- Ibrahim et al. 2007). Similarly, many HPLC, UPLC, cemic control which prevents the digestion of disac- and ion chromatographic methods are available for charides, oligosaccharides, and polysaccharides into the determination of 1-deoxynojirimycin as bulk and monosaccharides which can be absorbed by the body. in mulberry plants (Japan Intl. Research Center for As per the knowledge of the author, there was no Agricultural Sciences et al. 2010; Rudraprasad Reddy et analytical method proposed for Aza sugars with selec- al. 2014; Kimura et al. 2004; Rajana et al. 2016). tion. Many HPLC methods were there for the deter- The castanospermin and celgosivir are well biological mination of miglitol in drug products and drug active drugs for curing the different diseases (Budavari et substances (Balakumaran et al. 2016; Dhole et al. 1989; Hohenschutz et al. 1981; Saul et al. 1985; al. 2012; Chittora et al. 2009). Some of the HPLC Whitby et al. 2005; Durantel et al. 2009; Whitby et al. methods were available for the determination of 2004); these are highly polar polyhydroxyl Aza com- miglitol content in human plasma. The liquid chro- pounds. As per the author, no best analytical method matography is an available method for the identifica- can be determined for castanospermin and celgosivir as tion and quantification of miglitol in drug substance, single method. The present study is novel and also cap- drug product, and human plasma (Li et al. 2007; able of characterizing the process impurities and degrad- Nirogi et al. 2006; Wang et al. 2005). The unique ation impurities for castanospermin and celgosivir as separation techniques like electrophoresis, kinetic active pharmaceutical ingredients (International Confer- study instruments are used for the determination of ence on Harmonization (ICH) 2005; 1996; USP39 2016; miglitol in bulk drug substance (Cahours et al. 2002; Council of Europe 2015). The other polyhydroxyl Aza Fig. 6 HR-MS spectrum of castanospermin and its n-oxide impurity Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 5 of 16 sugars are characterized by high-resolution mass spec- Ion chromatography—optimization of chromatographic trometry and LC-MS/MS. conditions Metrohm ion chromatography instrument with MagIC Net 3.2 software was used for the total study. The Methods ion chromatography system was bought from Chemicals and reagents Metrohm, Herisau, Switzerland. The ion chromatog- 1-Deoxynojirimycin, miglitol, castanospermin, and celgosivir raphy system is equipped with 858 professional sam- are synthesized and purified from the process research and ple processor, 818 IC pump, sampling injector with a development of Custom Pharmaceutical Services of 20-μL loop, 882 compact IC plus with a cation sup- Dr. Reddy’s Laboratories Limited. Analytical reagent grade pressor, and a conductivity detector. Analysis of Aza tartaric acid, pyridine-2,6-dicarboxylic acid, sodium hydrox- sugars have been performed from the output signal, ide, hydrochloric acid, and hydrogen peroxide were monitored, and processed using the MagIC Net 3.2 purchased from Rankem (Mumbai, India). Millipore Milli Q version software on a Compaq computer (Digital purification system purchased from Bangalore, India. Photo Equipment Corp.). Dilutions were performed with stability chamber is with UV meter and master digital lux Hamilton Precision Pipettes (Bondaiz, Switzerland). meter purchased from Mack PharmaTech, India. Vacuum Chromatographic separation was performed on oven was purchased from Cintex, India. Metrosep C4 150/4.0 column at ambient temperature Fig. 7 Scan mode m/z of a celgosivir, b castanospermin, c n-oxide impurity of celgosivir, and d n-oxide impurity of castanospermin Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 6 of 16 with 4 mM tartaric acid and 1.5 mM pyridine-2,6-di- injection volume was 2.0 μL on Acquity TM binary solv- carboxylic acid mixed buffer and acetone (%v/v ent manager with waters TOF with LCT Premier XE. 90:10). The analysis was performed at 0.7 mL flow and 20-injection volume loop with conductivity LC-MS/MS—optimization of mass spectrometry detector. conditions LC-MS/MS study of castanospermin and celgosivir and their degradation impurities were recorded with High-resolution mass spectrometry—optimization of LC-MS G6410 QqQ instrument (Agilent Technolo- mass spectrometry conditions gies). The used condition was source temperatures at Water AQUATY UPLC-TOF instrument with LCT 325 °C, gas flow was 10 L/min, capillary voltage was Premier XE Mass Lynx TM software was used for the 4000 V, and the mass range was 100–800 a.m.u. The identification, characterization of process impurities, and heater temperatures were 100 °C for the both MS1 degradation impurities of castanospermin and celgosivir. and MS2 heaters. The EMV was 10 for mass spec- The mobile phase used for this study was 10 mM trometer. The collision gas was nitrogen for MS/MS ammonium acetate and acetonitrile (%v/v 50:50). The of APIs and degradation impurities. The different Fig. 8 MS/MS of celgosivir at a 10 eV collision energy, b 20 eV collision energy, c 30 eV collision energy, and d 50 eV collision energy Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 7 of 16 collision energies were used for the complete study of mark with diluent and cyclomix standards with vortex unknown degradation impurities. The LC condition cyclomixer. The solution was used for the ion chro- used were (% v/v) 25:75: 0.1% formic acid in water matography injection. and 0.1% formic acid in acetonitrile, the injection vol- ume for the present study of LC-MS/MS was 5.0 μL, and diluent was (%v/v: 10:90): acetonitrile and water. Specificity solution The MS/MS study was performed by using the flow The specificity of the ion chromatography method was 0.5 mL/min, run time 1.0 min, 0.25 mg/mL sample demonstrated with 1-deoxynojirimycin and miglitol concentration and union. along with castanospermin and celgosivir. The specificity standard solution used for this present study was Standard and sample solution preparation weighed and transferred about each 25 mg of 1- The Aza sugars’ standard solution was prepared by deoxynojirimycin, miglitol, castanospermin, and celgosi- weighing 25 mg of each catanospermin and celgosivir vir in a 100-mL volumetric flask contain 25 mL of water and transferring separately in to 100 mL volumetric and make up to the mark with diluent, cyclomix stan- flask containing 25 mL of water. Make up to the dards with vortex cyclomixer. Fig. 9 MS/MS of castanospermin at a 10 eV collision energy, b 20 eV collision energy, c 30 eV collision energy, and d 50 eV collision energy Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 8 of 16 Method validation of castanospermin and celgosivir by expected to be present. Typically, these might include ion chromatography impurities, degradants, matrix, etc. The specificity was The optimized ion chromatography method was vali- done for the no interference with other matrix and dated as per the ICH guidelines and validation of com- degradants along with studied active pharmaceutical in- pendial procedures as per USP general chapter 1225. gredients. The degradation study was done by acid and The validation parameters for the ion chromatographic base hydrolysis and oxidation; the degradation impurities method were specificity, precision, accuracy, linearity, were identified by using high-resolution mass spectrom- robustness, and ruggedness. etry and LC-MS/MS. Specificity Precision Specificity is the ability to assess unequivocally the ana- The precision of an analytical procedure expresses the lyte in the presence of components which may be closeness of agreement between a series of measurements Fig. 10 MS/MS of n-oxide impurity of celgosivir at a 10 eV collision energy, b 20 eV collision energy, c 30 eV collision energy, and d 50 eV collision energy Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 9 of 16 obtained from multiple sampling of the same homoge- Linearity neous sample under the prescribed conditions. Precision The linearity of an analytical procedure is its ability to may be considered at three levels: repeatability, intermedi- obtain test results which are directly proportional to the ate precision, and reproducibility. The precision was stud- concentration of analyte in the sample. The linearity of ied for the system suitability test and method precision, the ion chromatography method study for the 25 to 50% level and 150% level with respect to 100% level. 200% (w.r.t test concentration 0.25 mg/mL) has been Average, standard deviation, and variance for the six prep- studied, and correlation coefficient was calculated for arations were calculated for each parameter. both castanospermin and celgosivir. Accuracy Robustness It is also called trueness. The accuracy of an analytical The robustness of an analytical procedure is a measure procedure expresses the closeness of agreement between of its capacity to remain unaffected by small, but deliber- the value which is accepted either as a conventional true ate variations in method parameters, and provides an value or an accepted reference value and the value indication of its reliability during normal usage. To test found. The accuracy at 50, 100, and 150% levels were the robustness parameter, the precision and accuracy calculated, and average recovery was calculated at all were done by changing flow and buffer concentration, levels. and the %RSD were calculated. Fig. 11 MS/MS of n-oxide impurity of castanospermin at a 10 eV collision energy, b 20 eV collision energy, c 30 eV collision energy, and d 50 eV collision energy Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 10 of 16 Ruggedness chromatography. The peaks of castanospermin and celgosi- The ruggedness parameter is consisting of intermediate vir were eluted early, retained in C18 column with high- precision and repeatability. The intermediate precision concentration potassium buffer with sample concentration was performed by changing the different column and of 10 mg/mL, and they are less UV active in Fig. 1. Hence- different instrument in the same lab, the study was forth, the analytical method development has been started extended in different laboratories, and the results were by using ion chromatography technique with cation col- calculated and reported the %RSD for two labs. umn, conductivity detector, and ion chromatography com- patible buffers. In ion chromatography, the initial Solution and mobile phase stability development was started by using the universal column Solution stability is the stability for the same solution of with 3 mM nitric acid buffer and conductivity detector; the drug substance at different intervals of time with opti- Aza sugars were eluted as broad peaks with co-elution mized conditions. Mobile phase is stable at fresh prepar- (Fig. 2). The universal column has been replaced with ation in different intervals of time with the same mobile Metrosep C4 150/4.0 column with 3.0 mM nitric acid buf- phase along with other method parameters. fer: Acetone: 90:10 (%v/v), the peaks of 1-deoxynojirimycin, miglitol, and castanospermin were separated but not re- Results and discussion solved properly. Instead of Metrosep C4 150/4.0, the long Ion chromatography—method development and column (Metrosep C4 250/4.0) with the same conditions optimization like the previous trials were used for resolution of 1- The objective of the ion chromatographic method is to de- deoxynojirimycin, miglitol, and castanospermin but the velop the novel isocratic method for assay determination in profile of all peaks came late and peaks become broad and Aza sugars having polyhydroxyl compounds such as 1- no good resolution was observed between the 1- deoxynojirimycin, miglitol, castanospermin, and celgosivir. deoxynojirimycin, miglitol, and castanospermin (Fig. 3). The degradation study of castanospermin and celgosivir The weak organic buffer such as 4 mM tartaric acid, has been carried out by ion chromatography and 0.75 mM pyridine-2,6-dicarboxylic acid mixed buffer and characterization by high-resolution mass spectrometry and acetonitrile used (%v/v 90:10), but the resolution between LC-MS/MS. In initial stage, analytical method development 1-deoxynojirimycin and miglitol was less. The study was has been started with ultra-performance liquid continued with acetone instead of the acetonitrile; the Table 1 m/z values of castanospermin, celgosivir, and its oxidative impurities Name of product Molecular ion (m/z) Collison energy (eV) Fragment ions (m/z) Celgosivir 260.10 0 ND 260.10 10 242,172,154,136,116,72 260.10 20 242,172,154,136,126,116,98,80,72,56 260.10 30 172,154,136,126,116,108,98,80,72,56 260.10 50 136,126,116,108,98,80,72,56 Castanospermin 190.10 0 ND 190.10 10 172,154,136,86,72 190.10 20 172,154,136,126,112,98,86,80,72,58 190.10 30 172,154,136,118,112,100,86,69,56 260.10 50 136,112,93,82,68,56 Celgosivir 276.10 0 ND (Oxidative impurity) 276.10 10 206,158 276.10 20 206,171,158,124,102,71 276.10 30 206,171,158,145,114,86,68 276.10 50 158,112,98,86,68 Castanospermin 206.10 0 ND (Oxidative impurity) 206.10 10 172 206.10 20 188,172,154,145,116,98,82 206.10 30 188,172,154,145,128,116,98,86,68,57 206.10 50 106,98,86,68,57 ND Not detected Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 11 of 16 resolution increased more than acetonitrile trial, then the proton adduct, i.e., [M + H] + and the unknown degrad- concentration of tartaric acid increased from 4 to 5 mM ation peaks of castanospermin and celgosivir were identi- the all peaks came early with same conditions of above tri- fied as n-oxides of castanospermin and celgosivir. The als, all the peaks eluted early than previous profile and ob- mobile phase used for this study was 10 mM ammonium served the resolution also less. Finally, 4 mM tartaric acid acetate and acetonitrile (%v/v 50:50). The injection volume and 1.5 mM pyridine-2,6-dicarboxylic acid mixed buffer was 2.0 μL on Acquity ™ binary solvent manager with and acetone (%v/v 90:10) was used with 0.7 mL flow and waters TOF with LCT Premier XE. Instrument parameters 20-injection volume loop with conductivity detector and were polarity, ES+, Analyser, W-Mode, Capillary (V) Metrosep C4 150/4.0 column, and analysis was done at am- 1500.0, sample cone (V): 20.0, desolvation temp (°C):250.0, bient temperature (Fig. 4). The profile of all selected Aza source temp (°C):120.0, cone gas flow: 25.0, desolvation sugars and degradation impurities was well resolved. The flow: 250.0, ion guide one: 5.0, aperture 1 voltage: 5.0. ion final method was validated against the ICHQ2 (R1). energy(V): 29.0, aperture 2 voltage: 1.5, Hex pole DC volt- age:5.0, aperture 3 voltage: 10.0, acceleration (V): 90.0, Y High-resolution mass spectrometry—method focus (V): 68.0, steering (V): 2.1, tube lens (V): 30.0, attenu- development, optimization, identification, and ated: Z, focus (V): 85.9, TOF fight tube(V): 7200.0, reflec- characterization of unknown degradation impurities tron (V): 1800.0, pusher (V): 900.0, pusher offset voltage: The development of the ESI method was done with DIP 0.9, puller voltage: 752.0, puller offset (V): 0.0, MCP mass and LC-MS; the mass of 1-deoxynojirimycin, migli- detector (V): 2600, PusherCycleTimeAuto(60), PusherFre- tol, castanospermin, and celgosivir were observed with quency:16666.67, pusherwidthe: 400, Centroidthreshold: Fig. 12 Probable fragmentation pattern of celgosivir and n-oxide impurity Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 12 of 16 1.0, Minpoints: 2.0, Npmultiplier: 0.70, resolution: 9533.0, − 0.5 ppm with 1.5 DBE. Hence, the castanospermin perox- Lteff: 2225.0000, Veff: 7193.7285, trigger threshold (Mv): ide degradation impurity was proposed as n-oxide impurity 600.0000. Signal Threshold (mV) 60.0000, Data Threshold: of castanospermin (Fig. 6). 0.0000, DXC Temperature: 25.0, Scans in function: 277, Cycle time (secs): 0.410, Scan duration (secs):0.40, Inter LC-MS/MS—method development, optimization, scan delay (s):0.01, retention window (mins): 0.000 to identification, and characterization of unknown 10.000, Ionization mode: ES+, function type: TOF MS, degradation impurities Mass range: 105 to 1000 (Figs. 6 and 7). The MS/MS spectra of celgosivir and castanospermin and The formulae of all selected Aza sugars and its degrad- their peroxide degradation impurities are shown in Figs. 7 ation impurities were matching with theoretical mass num- and 8. At lower collision energies, i.e., 10 and 20 eV, the ber with less than 5.0 ppm error value. The celgosivir molecular ion peak at 260 and 190 m/z was observed for molecular formula (C12H22NO5: [M + H]) accuracy error celgosivir and castanospermin respectively (Figs. 8, 9, and ppm was − 4.6 ppm, and the celgosivir peroxide degrad- 10). At higher collision energies, i.e., 30 and 50 eV, the buta- ation impurity molecular formula (C12H22NO6: [M + H]) none group in celgosivir and –OH group in castanosper- was 0.4 ppm with 2.5 DBE. Hence, the celgosivir peroxide min were eliminated and observed 206 m/z for celgosivir degradation impurity was proposed as n-oxide impurity of and 172 m/z for castanospermin, further fragmented to celgosivir (Fig. 5). Similarly, the castanospermin molecular 158 m/z for the celgosivir (Fig. 8) and 154 m/z for casta- formula (C18H16NO4: [M + H]) accuracy error ppm was nospermin (Fig. 9). HR-MS data supported the fragmenta- − 4.7 ppm and the castanospermin peroxide degradation tion profile of celgosivir and castanospermin (Figs. 6 and 7). impurity molecular formula (C18H16NO5: [M + H]) was At the lower collision energies, the stable molecular ion Fig. 13 Probable fragmentation pattern of celgosivir and n-oxide impurity Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 13 of 16 Fig. 14 Selected Aza sugars product names, structures, chemical names, and empirical formulae peaks were at 276 and 206 m/z for n-oxides of celgosivir impurities (Table 1, Additional file 1). HR-MS data sup- and castanospermin respectively. At higher collision ener- ported the fragmentation profile of n-oxides of celgosivir gies, the stable molecular ion peaks were at 206 and and castanospermin and characterized as n-oxides of celgo- 188 m/z for n-oxides of celgosivir and castanospermin re- sivir and castanospermin (Figs. 12, 13, and 14; Table 2). spectively. The hydroxyl group loss stated that 206 and 188 m/z for celgosivir and castanospermin, respectively, Method validation for castanospermin and celgosivir by was observed. Further fragmentation gave continuous loss ion chromatography of hydroxyl group by using higher collision energies (Figs. 8, Specificity 9, 10, and 11). The fragmentation pattern difference ob- Chromatography obtained with mixer of castanosper- served was 16 mass number such as APIs and its n-oxide min and celgosivir shows no interference with other Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 14 of 16 Table 2 HR-MS pseudo fragmentation profile for formulae 1.2 million lux hours, visible light, 200 W/m UV confirm of the n-oxide impurity of castanospermin and celgosivir light, and thermal degradation at 105 °C. The degrad- Name of product Fragmentation Formulae ation peaks have been identified and characterized as n-oxides of castanospermin and celgosivir by high- Castanospermin 190.1070 [M + H] C H NO 8 16 4 resolution mass spectrometry and LC-MS/MS. The 172.0965 C H NO 8 14 3 peak purity was evaluated by high-resolution mass 154.0872 C H NO 8 12 2 spectrometry and LC-MS/MS. The mass balance is 136.0753 C H NO 8 10 demonstrated in Table 3. 112.0743 C H NO 6 10 n-Oxide impurity of CAS 206.1027 [M + H] C H NO 8 16 5 Precision (unknown impurity) The precision data obtained for the evaluated method 172.0968 C H NO 8 14 3 is demonstrated in Table 4. The precision at 50, 100, 154.0857 C H NO 8 12 2 and 150% were evaluated for castanospermin and cel- Celgosivir 260.1486[M + H] C H NO 12 22 5 gosivir in a mixer solution. The %RSD at (n =6) all 172.0972 C H NO levels were less than the 2.0% assuming acceptable 8 14 3 precision. 154.0834 C H NO 8 12 2 242.1272 C H NO 12 20 4 Accuracy Oxide impurity of CEL 276.1448 [M + H] C H NO 12 22 6 (unknown impurity) Accuracy was performed by means of recovery studies using the developed method. The percentage of 206.1008 C H NO 8 16 5 recoveries after spiking with additional mixer of casta- 158.0816 C H NO 7 12 3 nospermin and celgosivir was at all levels such as 50, 100, and 150% in the range of 98–102%, and the Aza sugars such as 1-deocynojirimycine, miglitol, and results are listed in Table 4. their degradants. During the degradation study, the n- oxide impurities of castanospermin and celgosivir were formed with 3% hydrogen peroxide (Table 3) Linearity and no interference with other cations. The n-oxide A linear relationship was presented between the concen- impurities of castanospermin and celgosivir were tration and peak area. The linearity concentration for identified by isolation at the column end and charac- the mixer of castanospermin and celgosivir was taken to terized by high-resolution mass spectrometry and LC- seven levels such as 25, 50, 75, 100, 125, 150, and 200%. MS/MS. Degradation study of castanospermin and The correlation coefficient value (r) and regression coef- celgosivir were performed by acid hydrolysis (0.5N ficient value (r) obtained for both was more than 0.999, HCl), base hydrolysis (0.5% Tri ethyl amine and 0.5N which explains the linearity of the method, and the re- NaOH), oxidation (3% H202), photo degradation at sults are listed in Table 4, (Plot 1 and Plot 2). Table 3 Summary of forced degradation results Stress condition Duration Assay of after forced degradation Assay of after forced degradation Content of major Remarks castanospermin (%w/w) of celgosivir (%w/w) degradant (%w/w) Acid hydrolysis 10 days 100 100 – No degradation products formed for both Base hydrolysis 0 h .0 100 100 of CAS formed No degradation from CIL after 0 h products formed for CAS Oxidation 6 h 79.2 90.0 20.8% n-oxide of CAS, n-oxides both 10% of n-oxide CIL sugars Thermal (105 °C) 7 days 100 100 – No degradation products formed for both UV light 200 W/m 100 100 – No degradation products formed for both Visible light 1.2 million 100 100 – No degradation lux hours products formed for both Rajana et al. Journal of Analytical Science and Technology (2018) 9:5 Page 15 of 16 Table 4 Summary of validation results method with less amount of the sample quantity was determined. The method was validated as per ICH Parameter Castanospermin Celgosivir Q2 (R1); the proposed ion chromatography method is Correlation coefficient (r) 1.000 0.999 found to be simple, sensitive, accurate, precise, spe- Intercept (c) − 0.01 − 0.01 cific and green chemistry type of analysis for diluent Slope (m) 0.1 0.03 and mobile phase preparation; it can be used for Method precision (%RSD) 1.1 0.7 intended purposes in drug substance and drug prod- Intermediate precision (%RSD) 3.2 3.1 uct. The n-oxide degradation impurities of castanos- Precision at 50% 1.2 1.7 permin and celgosivir under peroxide degradation were identified and characterized by using the high- Precision at 150% 0.4 1.9 resolution mass spectrometry and the LC-MS/MS. Accuracy at 50% (%recovery) 100.1 95.4 These novel techniques will help to improve the qual- Accuracy at 100% (%recovery) 99.7 99.7 ity ofthe drugs because the most ofAzasugars are Accuracy at 150% (%recovery) 97.0 82.3 using in diabetic research and dengue research. Precision at flow 0.6 mL/min (%RSD) 0.5 0.4 Precision at Flow 0.8 mL/min (%RSD) 1.1 0.7 Additional file low buffer strength (%RSD) 0.5 1.5 Additional file 1: Fragmentation pattern differences. (DOCX 761 kb) High buffer strength (%RSD) 2.4 0.5 Solution stability and mobile phase − 1.0 − 8.5 difference Acknowledgements The authors would like to thank Dr. Reddy’s Laboratories Ltd., With respect to initial (%recovery variation) Hyderabad, India, for providing the facilities to carry out this study. Cooperation from colleagues of Analytical Research and Development and the process research and development of Dr. Reddy’s Laboratories Robustness Ltd. is thankfully acknowledged. Robustness was performed by changing flow, i.e., from Authors’ contributions 10% to lower to actual flow and 10% higher flow from PM, JMB, KB, and DRD conceived and designed the experiments. NR and actual flow, and buffer concentration flow, i.e., the study BbD performed the experiments and wrote the manuscript. All authors read has been performed from 10% to lower concentration and approved the final manuscript. and 10% higher concentration from actual concentra- Competing interests tion. The evaluated parameters were performed for the The authors declare no that they have no competing interests. precision, %RSD were calculated and the results are listed in Table 4. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Ruggedness published maps and institutional affiliations. The %RSD values for intermediate precision and repeat- Author details ability reported were found to be less than 2% which Analytical Research and Development, Dr. Reddy’s Laboratories, Hyderabad, shows ruggedness of the ion chromatography method. 2 Telangana 500049, India. Department of Inorganic and Analytical Chemistry, The results of ruggedness parameters are listed in Table 4. Andhra University, Visakhapatnam, Andhra pradesh 530003, India. AU College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra pradesh 530003, India. Solution and mobile phase stability Solution stability was performed with same mixer of cas- Received: 26 September 2017 Accepted: 10 January 2018 tanospermin and celgosivir in active pharmaceutical in- gredient solution at 24 and 48 h. The mobile phase was References observed as stable in fresh mixer (for the assay of casta- Balakumaran K, Janagili M, Rajana N, Papureddy S, Anireddy J. 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"Journal of Analytical Science and Technology" – Springer Journals
Published: Dec 1, 2018
Keywords: Analytical Chemistry; Characterization and Evaluation of Materials; Monitoring/Environmental Analysis
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