Abstract
Stability-indicating High-Performance Thin-Layer Chromatography (HPTLC) method for simultaneous estimation of cefixime trihydrate and azithromycin dihydrate was developed. Both the drugs were subjected to different stress conditions recommended by International Conference on Harmonization (ICH) guideline Q1A (R2). Forced degradation was carried out for hydrolytic, oxidative, photolytic, and thermal degradation conditions. Cefixime was susceptible for degradation under all stress conditions showing four degradation products (CI–IV). However, azithromycin formed only one degradation product (AI) under acid hydrolysis. Aluminum plates precoated with silica gel 60F254 were used as the stationary phase while mixture of ethyl acetate–methanol–acetone–toluene–ammonia (1:5:7:0.5:0.5, v/v) was used as mobile phase. Detection wavelength used was 235 nm for CEFI and CI–IV. AZI and AI were detected by post development derivatization, spraying with sulfuric acid–ethanol (1:4, v/v) followed by heating at 100 °C for 5 min. Degradation products were isolated by preparative HPTLC and characterized by MS/MS. The developed method was validated for linearity, precision, accuracy, specificity, and robustness and has been successfully applied in the analysis of these drugs in tablet dosage form.
1. Introduction
Pharmaceutical drugs are combined many times to have a beneficial effect. Comparative stability of such combined drugs is necessary as it can affect the actual available concentrations of the drugs in the biological systems. Cefixime (CEFI) is chemically (6R, 7R)-7-[[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(carboxymethoxyimino) acetyl] amino]-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid with molecular formula C16H15N5O7S2 and molecular weight as 453.4496. It is a third generation oral cephalosporin antibiotic. Azithromycin (AZI) is chemically (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-β-d-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. Its molecular formula is C38H72N2O12 and molecular weight is 748.9845. It is a macrolide antibiotic. Both the drugs are official in Pharmacopeia (IP) 2014 [1] and United States Pharmacopeia (USP) 2014 [2]. Combination of CEFI and AZI is available as in the form of tablets (Omnicef-AZ®) and is widely used in treatment of acute otitis media, non-streptococcal bacterial pharyngitis.
Literature survey indicated availability of stability-indicating high-performance liquid chromatography (HPLC) [3–23], high-performance thin-layer chromatography (HPTLC) [24–26], and spectrophotometric [27–29] methods for CEFI and AZI alone and as a combination with other pharmaceutical drugs. However, there is only one stability-indicating reversed-phase (RP)-HPLC method for simultaneous estimation of CEFI and AZI [30]. Hence, it was aimed to develop and validate simple stability-indicating HPTLC method and also to characterize degradation products.
2. Experimental
2.1 Materials and Instruments
Pure drugs cefixime and azithromycin were received as gift samples along with certificates of analysis from Maxim Pharmaceuticals Pvt. Ltd., Pune, India and Alembic Ltd, Vadodara, India, respectively. All used solvents and reagents were of analytical grade. Methanol was purchased from Merck Specialities Pvt. Ltd., Mumbai. Other reagents like triethylamine, hydrochloric acid, sodium hydroxide, and hydrogen peroxide were procured from Loba Chemie Pvt. Ltd., Mumbai. TLC plates precoated with Silica Gel 60F254 (20 cm × 10 cm, 200 μm thickness, E. Merck, Germany) were used as stationery phase.
HPTLC System (Camag, Muttenz, Switzerland) comprising of Linomat 5 applicator, Automatic TLC Sampler 4 (ATS4), TLC scanner 3, and winCATS software V 1.4.2 was used for chromatographic separation. High-resolution liquid chromatography/mass spectrometry (HR–LC/MS) system (Agilent Technologies, Inc, United States) consisting of 1290 Infinity UHPLC System, 1260 infinity Nano HPLC with Chipcube, 6550 iFunnel Q-TOFs and LC–MS/MS (Agilent Technologies, Inc., United States) system 6310 with Ion Trap Agilent LC, an atmospheric pressure ion source, ion trap mass spectrometer (model 6330), and ChemStation-LC control software was used for characterization of degradation products.
2.2 Preparation of Analyte Stock Solutions
Accurately weighed quantity (10 mg) of both drugs (CEFI and AZI) was separately transferred to a 10 mL volumetric flask and dissolved in 5 mL of methanol. Methanol was carefully added up to the mark to these volumetric flasks after the drugs were dissolved to get a stock solution of strength 1 mg/mL.
2.3 Selection of Analytical Wavelength
A quantity of 0.1, 0.2, 0.3, 0.4, and 0.5 mL of each of the stock solutions was transferred to ten different 10 mL volumetric flasks. The volume was then made up by adding methanol. This generated the solutions of concentrations 10, 20, 30, 40, and 50 μg/mL. Each of these samples was scanned over the range 200 to 400 nm to determine the wavelength of maximum absorbance.
2.4 Stress Studies
Hydrolytic degradation of both the drugs was carried out under acidic, alkaline, and neutral conditions separately by mixing 1 mL of drug stock solution with 1 mL each of HCl (1 M, 0.1 M, 0.01 M), NaOH (1 M, 0.1 M, 0.01 M), and water and maintaining them at various temperatures for 0–24 h. After the required exposure, samples were neutralized by adding solution of acid or base of the same strength as that of the stressor. For studying oxidative stress, 1 mL of each of the stock solutions was treated separately with 1 mL of 3%, 15%, and 30% of H2O2 at room temperature (RT) for 24 h. Effect of dry heat (thermal degradation) and light (photo degradation) was studied on solid state. In case of thermal degradation, the drug powder contained in sealed glass ampoule was heated in an oven at 60 °C/80 °C/100 °C/200 °C for a period of 8 days. Control sample was maintained in the same way at room temperature.
During photodegradation, solid drug powder was exposed to fluorescent light (1.25 million lux h) and ultraviolet (UV) light (200 Wh/m2) in a photostability chamber along with control samples (covered with aluminum foil).
2.5 Preparation of Samples for HPTLC
All degradation samples after requisite exposure to stress conditions were diluted with methanol to obtain concentration of individual drug as 100 μg/mL. Samples stressed in solid state were weighed appropriately and diluted with methanol to produce 100 μg/mL of both the drugs. A quantity of 10 μL of degraded sample of CEFI and 1 μL of degraded sample of AZI were applied on precoated HPTLC plates to get a concentration of 1000 ng/band and 100 ng/band of CEFI and AZI, respectively. The results were obtained by comparing samples subjected to stress treatment with appropriate controls (std. untreated). Peak area of drug after degradation was compared with the peak area of standard to know % degradation. Conditions were optimized with respect to strength of stressor, time, and temperature to get 5%–30% degradation of individual drugs. Optimized stress conditions are shown in Table 1.
Stress conditions for optimum degradation of CEFI and AZI
| Stress condition | Exposure condition (stressor, temperature, duration of exposure) | % Drug degradation | ||
|---|---|---|---|---|
| CEFI | AZI | CEFI | AZI | |
| Acid hydrolysis | 0.5 M HCl, RT, 0.5 h | 0.5 M HCl, RT, 1 h | 15.65 | 16.45 |
| Alkaline hydrolysis | 0.5 M, NaOH, RT, 30 min | 27.56 | 20.76 | |
| Neutral hydrolysis | H2O, 2 h | 21.39 | 24.01 | |
| Oxidation | 30% H2O2 RT, 1 h | 3% H2O2, RT 1 h | 21.92 | 22.88 |
| Photolysis | Fluorescent light 1.2 million lux h and UV light 200 Wh/m2 | 13.87 | 11.34 | |
| Thermal | 100 °C, 1 h | 200 °C, 2 h | 26.29 | 19.38 |
2.6 Development of Stability-Indicating Analytical Method (SIAM)
CEFI is comparatively non-polar, and AZI is extremely polar drug because of presence of hydroxyls groups, and sugar moieties in the structure. Initially, plates were spotted with 10 μL of stock solution and developed by linear ascending development method using different solvents like toluene, hexane, methanol, chloroform, dichloromethane, ethyl acetate, acetone, and acetonitrile. Based on the results obtained, binary and ternary mixtures of solvents were tried to achieve optimum resolution between drugs as well as degradation products and good peak shape.
The samples were spotted in the form of bands of width 8 mm with a CAMAG microliter syringe on precoated silica gel aluminum plate 60 F254 using a CAMAG Linomat V (Switzerland) sample applicator. A constant application rate of 100 nL/s was employed, and space between two bands was 14 mm. The chamber saturation time for mobile phase was 20 min. Chromatogram was developed in a CAMAG twin-trough chamber using a linear ascending technique. The length of chromatogram run was 8 cm. Subsequent to the development, the TLC plates were dried in a current of air. Densitometric analysis was performed on a CAMAG TLC Scanner III. Due to lack of chromophore in the structure of AZI, different spraying reagents were tried to visualize the parent drug and its degradation products. The slit dimension was kept at 6 mm × 45 mm, and 20 mm/s scanning speed was employed.
2.7 Method Validation
For linearity, from the stock solution different aliquots were spotted to obtain concentrations such as 500, 1000, 1500, 2000, and 2500 ng/band for CEFI and 50, 100, 150, 200 and 250 ng/band for AZI. The solutions were prepared in triplicate, and the peak area versus concentration data was processed by least-square linear regression analysis. The intra- and inter-day variation for the determination of CEFI and AZI were carried out in triplicates at three different concentrations, 800, 1000, and 1200 ng/band for CEFI and 100, 150, 200 ng/band for AZI. Intra-day variations were determined on the same day at different time intervals (morning, afternoon, evening), while inter-day variations were determined by analyzing the samples exactly at the same time under identical conditions for three consecutive days.
For recovery, the mixture of degradation samples was spiked with 80, 100, and 120 μg/mL of the standard CEFI and 100, 150, 200 μg/mL of the standard AZI. A quantity of 10 μL of the CEFI and 1 μL of the AZI solutions was applied on plate so as to get concentrations of 800, 1000, and 1200 ng/band for CEFI and 100, 150, and 200 ng/band for AZI. Plates were developed by the method described in Section 3.2. The experiment was conducted in triplicate. Percent recoveries were calculated.
The specificity of the method was ascertained by analyzing standard drugs and degraded samples. The band for CEFI and AZI in degraded samples was confirmed by comparing the RF values and UV spectrum of the band with that of the standard. Peak purities of drugs and degradation products were assessed by comparing the spectra at three different levels, viz., peak start, peak apex, and peak end positions. Robustness was checked by introducing small and deliberate changes in the mobile phase composition, absorbance wavelength, and saturation time. Effects of such variations on the results were examined. Mobile phases having different amount of ammonia (0.5 ± 0.1, v/v), absorbance wavelengths (235, 530 ± 2 nm), and 20 ± 5 min saturation times were tried at concentration levels of 1000 and 150 ng/band of CEFI and AZI, respectively, in triplicate.
2.8 Analysis of the Marketed Formulation
Twenty tablets of Omnicef-AZ® containing 200 mg of CEFI and 250 mg of AZI per tablet were weighed separately; their mean weight was determined, and tablets were finely powdered. Tablet powder equivalent to 50 mg CEFI was transferred into a 50 mL volumetric flask containing 40 mL methanol, sonicated for 20 min, and diluted to 50 mL with methanol. The resulting solution was filtered through Whatman filter paper no. 40 and analyzed chromatographically after appropriate dilution.
2.9 Kinetics of Degradation
All degradations were performed at initial concentration of 1 mg/mL, that is 0.0022 M for CEFI and 0.00134 M for AZI. Acid hydrolysis (1 M HCl), alkaline hydrolysis (1 M NaOH), oxidation (3% H2O2, i.e., 0.012 M), thermal degradation (80 °C for CEFI and 200 °C for AZI), and photolysis (254 nm) as explained under Section 2.4 were performed. Order of a particular reaction was found by graphical method, and corresponding rate constants were calculated.
2.10 Preparative Isolation and Characterization of Degradation Products
Thermal degradation sample of CEFI and acid degradation sample of AZI (400 μL) was applied as 180 mm band on 20 × 10 cm plate. Plate was developed in the optimized mobile phase. Drug and degradation product bands were marked with pencil, and plates were cut carefully to separate different bands. Individual bands were cut into strips and sonicated with methanol for extraction of degradation products. Methanol fractions were concentrated and evaporated to obtain solid residue which was subjected to mass fragmentation studies. High-resolution mass spectroscopic studies gave accuracy of the masses up to 4 decimals and MSn studies helped in locating origin of each fragment.
3. Results and Discussion
3.1 Selection of Wavelength for Analysis
The UV spectrum of CEFI and AZI solution (10 μg/mL) prepared using methanol was recorded. CEFI showed wavelength maximum at 235 nm, so it was selected as a detection wavelength for it. AZI lacks chromophore in UV region; hence, it was derivatized by spraying plate with sulfuric acid: ethanol (1:4, v/v) followed by heating at 100 °C for 5 min and detected at 530 nm.
3.2 Selection of Chromatographic System
The TLC procedure was optimized to quantify CEFI and AZI from bulk and degraded samples. Both the pure drugs were spotted on TLC plates and run in different solvent systems. Initially, binary systems were tried. Based on the results obtained ternary systems were developed. Two drugs were retaining at two corners of the plate because of their opposite polarities. Later, quaternary system containing ethyl acetate, methanol, acetone, and toluene gave good resolution, but typical peak nature was missing. Finally, introduction of ammonia gave sharp and well-defined bands at acceptable RF values. Chamber was saturated with mobile phase 20 min at room temperature. The optimized HPTLC method for simultaneous estimation of CEFI and AZI was ethyl acetate–methanol–acetone–toluene–ammonia (1:5:7:0.5:0.5, v/v).
3.3 Development of SIAM
The densitograms of the samples treated with acid, alkali, neutral, hydrogen peroxide, dry heat, and UV light showed well-separated bands of pure CEFI and AZI as well as some additional peaks at different RF values. The bands of the degraded products were well-resolved from the drug bands. Results are shown in Figures 1 and 2.
Densitograms. (a) Standard CEFI (RF = 0.28) and AZI (RF = 0.67); (b) CEFI — thermal degradation, (c) CEFI — oxidation, (d) CEFI — photolysis, (e) AZI — acid hydrolysis
Citation: Acta Chromatographica Acta Chromatographica 30, 4; 10.1556/1326.2017.00199
Photodocumentation of developed plate with bands of (a) CEFI and degradation products at 254 nm, and (b) AZI and its degradation products at 530 nm
Citation: Acta Chromatographica Acta Chromatographica 30, 4; 10.1556/1326.2017.00199
The densitograms of the acid, alkaline, and neutral hydrolyzed sample for CEFI showed one common peak (CI) at 0.17. This CI was found in all degradation conditions of CEFI. The densitograms of the CEFI upon oxidation showed an additional peak (CIII) at RF value of 0.42. Photodegraded sample of CEFI showed presence of CIV at RF value 0.52 along with CI and CIII. Thermal degraded sample showed a total of 4 peaks corresponding CI, CIII, CIV, and additionally CII at RF value 0.37. AZI showed only one additional peak (AI) at RF value of 0.6 upon acid and neutral hydrolysis. Kinetic studies (Table 2) indicated first-order reaction for acid/alkaline hydrolysis and oxidation for both the drugs except for oxidation of CEFI. However, owing to large excess of reagents as compared to the parent drug, these seem to be actually of second order, i.e., pseudo first orders. Thermal degradation and photolysis tend to follow zero order kinetics. Velocity wise, oxidation and thermal degradation of CEFI are of the same rate. Photolysis of CEFI seems to be the slowest among all, even 1/3rd of that of photolysis of AZI. Acid hydrolysis of CEFI is faster, having a rate of reaction almost twice than that of AZI as well as alkaline hydrolysis of both the drugs.
Kinetics of degradation
| Degradation condition | Order of reaction | Rate constant |
|---|---|---|
| CEFI | ||
| Acid hydrolysis | First order | 0.500 h−1 |
| Alkaline hydrolysis | First order | 0.263 h−1 |
| Oxidation | Zero order | 0.0004 M h |
| Photolysis | Zero order | 0.00006 M h |
| Thermal degradation | Zero order | 0.0004 M h |
| CEFI | ||
| Acid hydrolysis | First order | 0.209 h−1 |
| Alkaline hydrolysis | First order | 0.228 h−1 |
| Oxidation | First order | 0.2761 h−1 |
| Photolysis | Zero order | 0.0002 M h |
| Thermal degradation | Zero order | 0.0001 M h |
3.4 Method Validation
Summary of validation parameters is shown in Table 3a and b. The peak purity of CEFI and AZI was assessed by comparing the spectra at peak start, peak apex, and peak end positions proving specificity of the method.
Summary of validation parameters
| Parameter | Result | |||
|---|---|---|---|---|
| CEFI | AZI | |||
| Linearity | Equation of line | y = 2.1614x + 1002 | y = 27.89x − 313 | |
| r 2 | 0.999 | 0.998 | ||
| Range (ng/band) | 500–2500 | 50–250 | ||
| Sensitivity | LOD (ng/band) | 58 | 3.25 | |
| LOQ (ng/band) | 175 | 9.84 | ||
| Precision | Actual concentration | Intra-day predicted concentration, mean ± % RSD | ||
| CEFI | AZI | CEFI | AZI | |
| 800 1000 1200 | 100 150 200 | 799.29 ± 034 1000.58 ± 0.46 1201.38 ± 0.42 | 99.52 ± 0.31 149.67 ± 0.48 200.63 ± 0.50 | |
| Actual concentration | Inter-day predicted concentration, mean ± % RSD | |||
| CEFI | AZI | CEFI | AZI | |
| 800 1000 1200 | 100 150 200 | 798.55 ± 0.95 999.04 ± 0.93 1199.06 ± 0.72 | 98.94 ± 0.73 148.81 ± 0.99 199.91 ± 0.81 | |
| Accuracy | Spiked concentration | Recovery (%), mean ± % RSD | ||
| CEFI | AZI | CEFI | AZI | |
| 800 1000 1200 | 100 150 200 | 98.85 ± 1.39 99.85 ± 0.20 101.09 ± 0.82 | 99.52 ± 0.34 100.66 ± 0.54 100.08 ± 0.37 | |
| Robustness | (% RSD) | Less than 2 | Less than 2 | |
Specificity of the proposed method for CEFI and AZI
| Drug/degradation products | Retardation factor (RF) | Peak purity (>0.999) |
|---|---|---|
| CI | 0.17 | 0.9993 |
| CII | 0.37 | 0.9996 |
| CIII | 0.42 | 0.9994 |
| CIV | 0.52 | 0.9996 |
| CEFI | 0.28 | 0.9997 |
| AI | 0.60 | 0.9994 |
| AZI | 0.67 | 0.9996 |
Assay of marketed formulation
| Tablet formulation | Label claim (mg/tablet) | Amount of drug estimated (mg) | % of label claim mean ± RSD (%) |
|---|---|---|---|
| Omnicef-AZ® | 200 mg, CEFI 250 mg, AZI | 200.48 | 100.24 ± 0.26 |
| 249.10 | 99.64 ± 0.54 |
Summary of HR–LC–MS/MS data of CEFI and its DPs
| Compound | EM | Best possible MF | TM | Difference | Error in mmu | Major fragments | Error in mmu | Best possible MF | RDB | |
|---|---|---|---|---|---|---|---|---|---|---|
| EM | TM | |||||||||
| CEFI | 454.0431 | C16H16N5O7S2+ | 454.0486 | −0.0055 | −5.5 | 328.0559 | 328.0598 | −3.9 | C12H14N3O6S+ | 7.5 |
| 284.025 | 284.0336 | −8.6 | C10H10N3O5S+ | 7.5 | ||||||
| 241.0353 | 241.0278 | 7.5 | C9H9N2O4S+ | 6.5 | ||||||
| 210.0177 | 210.0219 | −4.2 | C9H8NO3S+ | 6.5 | ||||||
| 152.0144 | 152.0165 | −2.1 | C7H6NOS+ | 5.5 | ||||||
| 126.0398 | 126.0372 | 2.6 | C6H8NS+ | 3.5 | ||||||
| 102.0393 | 102.0372 | 2.1 | C4H8NS+ | 1.5 | ||||||
| CEFI DP I | 398.0564 | C14H16N5O5S2+ | 398.0587 | −0.0023 | −2.3 | 381.0344 | 381.0322 | 2.2 | C14H13N4O5S2+ | 10.5 |
| 370.0268 | 370.0274 | −0.6 | C12H12N5O5S2+ | 9.5 | ||||||
| 352.0555 | 352.0533 | 2.2 | C13H14N5O3S2+ | 9.5 | ||||||
| 298.0477 | 298.0492 | −1.5 | C11H12N3O5S+ | 7.5 | ||||||
| 271.0312 | 271.0383 | −7.1 | C10H11N2O5S+ | 6.5 | ||||||
| 243.0424 | 243.0434 | −1 | C9H11N2O4S+ | 5.5 | ||||||
| 228.0369 | 228.0325 | 4.4 | C9H10NO4S+ | 5.5 | ||||||
| CEFI DP II | 227.0415 | C9H11N2O3S+ | 227.0485 | −0.007 | −7 | 210.0256 | 210.0219 | 3.7 | C9H8NO3S+ | 6.5 |
| 199.0102 | 199.0172 | −7 | C7H7N2O3S+ | 5.5 | ||||||
| 181.0411 | 181.043 | −1.9 | C8H9N2OS+ | 5.5 | ||||||
| CEFI DP III | 370.065 | C13H16N5O4S2+ | 370.0638 | 0.0012 | 1.2 | 353.0399 | 353.0373 | 2.6 | C13H13N4O4S2+ | 9.5 |
| 352.0511 | 352.0533 | −2.2 | C13H14N5O3S2+ | 9.5 | ||||||
| 324.0535 | 324.0583 | −4.8 | C12H14N5O2S2+ | 8.5 | ||||||
| 270.0523 | 270.0543 | −2 | C10H12N3O4S+ | 6.5 | ||||||
| 243.0448 | 243.0434 | 1.4 | C9H11N2O4S+ | 5.5 | ||||||
| 231.0422 | 231.0434 | −1.2 | C8H11N2O4S+ | 4.5 | ||||||
| 214.0143 | 214.0169 | −2.6 | C7H9N4O2S+ | 4 | ||||||
| CEFI DP IV | 428.0667 | C15H18N5O6S2+ | 428.0693 | −0.0026 | −2.6 | 410.0525 | 410.0587 | −6.2 | C15H16N5O5S2+ | 10.5 |
| 384.0781 | 384.0795 | −1.4 | C14H18N5O4S2+ | 8.5 | ||||||
| 213.0491 | 213.0441 | 5 | C7H9N4O2S+ | 5.5 | ||||||
| 115.0511 | 115.0502 | 0.9 | C4H7N2O2+ | 1 | ||||||
| AZI | 749.5105 | C38H73N2O12+ | 749.5158 | −0.0053 | −5.3 | 591.4174 | 591.4215 | −4.1 | C30H59N2O9+ | 2.5 |
| 434.3077 | 434.3112 | −3.5 | C22H44NO7+ | 1.5 | ||||||
| 158.1164 | 158.1176 | −1.2 | C8H16NO2+ | 5 | ||||||
| 116.1063 | 116.107 | −0.7 | C6H14NO+ | 0.5 | ||||||
| 83.0487 | 83.0491 | −0.4 | C5H7O+ | 2.5 | ||||||
| AZI DPI | 591.4187 | C30H59N2O9+ | 591.4215 | −0.0041 | −4.1 | 416.3248 | 416.3007 | 24.1 | C22H42NO6+ | 2.5 |
| 358.2500 | 358.2588 | −8.8 | C19H36NO5+ | 2.5 | ||||||
| 300.2158 | 300.2169 | −1.1 | C16H30NO4+ | 2.5 | ||||||
| 282.2128 | 282.2064 | 6.4 | C16H28NO3+ | 3.5 | ||||||
| 238.1896 | 238.1802 | 9.4 | C14H24NO2+ | 3.5 | ||||||
| 224.1606 | 224.1645 | −3.9 | C13H22NO2+ | 3.5 | ||||||
| 198.1494 | 198.1489 | 0.5 | C11H20NO2+ | 2.5 | ||||||
| 167.1075 | 167.1067 | 0.8 | C10H15O2+ | 3.5 | ||||||
EM: experimental mass, MF: molecular formula, TM: theoretical mass, RDB: ring plus double bond, CEFI: cefixime, AZI: azithromycin, DP: degradation product
MSn data for CEFI and AZI
| MSn stage | Precursor ion (m/z) | Product ion (m/z) |
|---|---|---|
| CEFI | ||
| MS2 | 454 | 334, 328, 318, 305, 284, 272, 257, 241, 226, 210, 181, 165, 154 |
| MS3 | 284 | 257, 241, 227, 210, 194, 181, 170, 165, 126, 116, 98 |
| MS4 | 241 | 213, 181, 165, 156, 137, 133, 126, 110 |
| AZI | ||
| MS2 | 749 | 591, 573, 434 |
| MS3 | 591 | 573, 434, 416, 398 |
| MS4 | 434 | 416, 376, 358, 341, 318, 300, 282, 275, 238, 224, 203, 198, 186, 173, 167, 155, 142 |
The linear regression data for the calibration plots as shown in Table 3a illustrate a good linear relationship over a concentration range of 500–2500 ng/band CEFI and 50–250 ng/band for AZI with respect to the peak area.
The measurement of the peak area at three different concentration levels showed low values of the relative standard deviation (% RSD) for inter and intra-day variation, which suggested an excellent precision of the proposed method. When degradation samples were spiked with pure drug, the method showed recovery between 98.85%–101.09% and 99.52%–100.66% for CEFI and AZI, respectively.
The % RSD of the peak areas was calculated after small deliberate changes in mobile phase composition, absorbance wavelength, and duration of chamber saturation to realize robustness.
3.5 Analysis of the Marketed Formulation
The drug content was found to be between 98 and 102% in the formulation tested. The low % RSD value indicated the suitability of this method for routine analysis of title ingredients in pharmaceutical dosage form. Results are shown in Table 4.
3.6 Isolation and Characterization of Degradation Products
Four degradation products for CEFI and 01 degradation product for AZI were isolated by preparative HPTLC and subjected to tandem mass spectrometry (MS/MS) studies for characterization. β-Lactam ring in cefixime is susceptible to ring opening under all conditions. Based on fragmentation pattern obtained, for CEFI, structures of CI (R)-2-((R)-(2-(2-aminothiazol- 4-yl)-2-iminoacetamido) (carboxy)methyl)-5-vinyl-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid, CII (6R,7R)-7-amino-8-oxo-3-vinyl-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid, CIII (R)-2-(2-(2-aminothiazol-4-yl)-2-iminoethylamino)-2-((R)- 7-oxo-2,4,5,7-tetrahydro-1H-furo[3,4-d][1,3]thiazin-2-yl)acetic acid, and CIV (E)-2-((2-(2-aminothiazol-4-yl)-2-(carboxymethoxyimino)acetamido)methyl)-5-vinyl-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid were confirmed.
Formation of CI involves opening of β-lactam ring and loss of carboxy methoxy side chain on imine. Formation of CII is because of elimination of entire (Z)-2-(2-amino-4-thiazolyl)-2-(carboxymethoxyimino) acetyl moiety at C-7. It is nothing but 7-amino-3-vinyl-3-cephem-4-carboxylic acid which is popular starting material for commercial synthesis of cefixime. Formation of CIII might be from CI with further epoxidation at vinyl group at C-3. CIV involves opening of β-lactam ring with unusual loss of carbonyl group (C6) of the β-lactam bond. One such compound is an official impurity in British Pharmacopoeia (BP).
Table 5 summarizes data about major fragments of CEFI and 4 degradation products. AZI was found to degrade to AI ((2R,3S,4R,5R,8S,10R,12R,13S,14R)-10-((2S,3S,6S)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yloxy)-2-ethyl-3,4,9,13-tetrahydroxy-3,5,6,8,9,12,14-heptamethyl-1-oxa-6-azacyclopentadecan-15-one). AZI contains two sugar moieties. One is an amino sugar, desosamine, while the other is a neutral sugar, cladinose. Desosamine resists acid hydrolysis much more than that of the cladinose; hence, AZI looses cladinose to form AI commonly known as azithralosamine. This compound has been used after derivatization as azithralosamine acetate in gas chromatography–mass spectrometry (GC–MS) detection of AZI in biological fluids [31]. Table 6 indicates data of MSn studies. Structures of all degradation products are presented in Figure 3.
Proposed pathways for degradation of (a) CEFI to DPs I–IV and (b) AZI to AI
Citation: Acta Chromatographica Acta Chromatographica 30, 4; 10.1556/1326.2017.00199
4. Conclusion
Stability-indicating HPTLC method was developed and validated; degradation products were identified, isolated by preparative HPTLC, and characterized for cefixime and azithromycin. Comprehensive mass fragmentation profile was established for both the drugs and their degradation products.
Both the drugs were susceptible to all kinds of degradation. CEFI was more prone for acid, alkaline, and thermal degradations than AZI, while it was stable comparatively for oxidation. For neutral hydrolysis and photolytic stress, both drugs showed similar degradation behavior quantitatively. The developed method can be used for estimation of these drugs from bulk, oral solid dosage forms and stability samples in the industry as their degradation products are well resolved from the drug as well as from each other. Identified degradation products can be used to synthesize reference standards as well as to monitor their presence in the stability samples.
Acknowledgements
Authors are thankful to Sinhgad College of Pharmacy (Pune, India), Anchrom Enterprises (Mumbai, India), and Sophisticated Analytical Instrument Facility (IIT Bombay, India) for providing necessary facilities to carry out the work.
References
- 1.↑
Indian Pharmacopoeia Vol. II, Government of India, The Controller of Publications: Delhi, 2014, pp. 1118–1305.
- 2.↑
United States Pharmacopoeia 38, National Formulary 33 United States Pharmacopoeial Convention: Rockville, 2014, pp. 395–432.
- 4.
Goswami J. A. ; Shah N. J. Indo Am. J. Pharm. Res. 2015, 5, 2021.
- 5.
Rathod L. ; Suvarna V.; Shinde N. Int. J. Pharm. Sci. Res. 2015, 43, 4886.
- 6.
Joshi M. P. ; Gamit K. G.; Parmar V. K.; Raval P. P. World J. Pharm. Pharm. Sci. 2014, 3, 1259.
- 8.
Badmanaban R. ; Patel U. M.; Patel A. S.; Patel C. N. Int. J. Pharm. Res. Bio-Sci. 2014, 3, 326.
- 9.
Hassan N. Y. ; Mostafa N. M.; Abdel-fattah L.; Weshahy S. A.; Boltia S. A. Aust. J. Basic & Appl. Sci. 2013, 7, 285.
- 10.
Talebpour Z. ; Pourabdollahi H.; Rafati H.; Abdollahpour A.; Bashour Y.; Aboul-Enein H. Y. Sci. Pharm. 2013, 81, 493.
- 11.
Adam E. H. K. ; Saeed A. E. M.; Barakat I. E. Int. J. Pharm. Sci. Res. 2012, 3, 469.
- 12.
Adam E. H. K. ; Saeed A. E. M.; Barakat I. E. Pharma Chem. 2011, 3, 197.
- 13.
Gandhi S. P. ; Rajput S. J. Indian J. Pharm. Sci. 2009, 71, 438.
- 14.
Namiki Y. ; Tanabe T.; Kobayashi T.; Tanabe J.; Okimura Y.; Koda S.; Morimoto Y. J. Pharm. Sci. 1987, 76, 208.
- 15.
Subbareddy P. ; Divakar T. E. Am. J. PharmTech Res. 2015, 5, 653–662.
- 16.
Ghode P. D. ; Pawar S. P. Int. J. Adv. Pharm. Anal. 2015, 5, 17.
- 17.
Krishna V. R. ; Krishna K. B. M.; Babu B. H. Indian J. Pharm. Sci. Res. 2014, 4, 176.
- 18.
Sudheer M. ; Rao A. B. N. N.; Theja D. H. H.; Prakash M. S.; Ramalingam P.; Mohan A. M. Pharm. Lett. 2012, 4, 803.
- 19.
El-Gindy A. ; Attia K. A.; Nassar M. W.; Al Abasawi N. M.; Al-Shabrawi M. J. AOAC Int. 2011, 94, 513.
- 20.
Al-Rimawi F. ; Kharoaf M. J. Chromatogr. Sci. 2010, 48, 86.
- 21.
Singh S. K. ; Prakash D.; Mani T. T. Int. J. ChemTech Res. 2010, 2, 1939.
- 22.
Zhang Y. ; Liu X.; Cui Y.; Huang H.; Chi N.; Tang X. Chromatographia 2009, 70, 67.
- 25.
Khedr A ; Sheha M. J. Chromatogr. Sci. 2003, 41, 10.
- 26.↑
Singh B. ; Parwate D.; Srivastava S.; Shukla S. J. Planar Chromatogr. – Mod. TLC 2011, 24, 524.
- 27.↑
Mostafa N. M. ; Abdel-Fattah L.; Weshahy S.A.; Hassan N.Y.; Boltia S.A. J. AOAC Int. 2015, 98, 35.
- 28.
Naveed S. ; Waheed N.; Nazeer S. Int. Res. J. Pharm. Sci. 2014, 5, 23.
- 30.↑
Ramesh M. ; Kanaka D. M.; Sravani A.; Snehalatha T.; Thimmareddy D. Asian J. Res. Chem. 2012, 5, 1067.
