Reactions between active drug substances and excipients are of interest in the drug formulation process should be checked for the interactions during the storage conditions. Some excipients react with certain chemical groups in drug substances which will form new impurities in the finished product formulations. In the present paper transesterification reaction of methylphenidate with glycerin to form different structural isomeric products was described. These impurities identified in forced degradation studies, excipient compatibility studies and stability analysis of the finished product. Stability samples were analyzed and observed that about ~0.6% of the Methylphenidate content was transformed into methylphenidate-glycerin isomers within 3 Months at 40°C/75% RH and 18 Months at 25°C/60% RH conditions. Analysis of two lots of marketed preparations having expiry dates in 2012 and 2013 showed content of the Methylphenidate esters corresponding to ~0.6% of the declared Methylphenidate content. The samples of this impurity were investigated by HPLC, UPLC-MS/MS to generate the mechanism of the impurity formation.
Methylphenidate (
Methylphenidate oral solution shows two unknown impurities in the related compound method which is more than the identification threshold as per the ICH guidelines. These impurities identified in forced degradation studies, excipient compatibility studies and stability analysis of the finished product. Manufacturer’s impurities and chemical instabilities were checked and identified that these impurities formed due to the drug excipient interaction of the Methylphenidate with glycerin. The objective of the present research article is to give a procedural identification of these unknown peaks and its formation, stability and toxicity in the Methylphenidate oral solution using HPLC, LC-MS/MS. The formation of these impurities is less than one percent, so that the isolation and characterization of these impurities was not feasible. Analysis of the stability samples, drug-excipient compatibility samples and commercially available marketed solutions were performed by LC-MS/MS and HPLC and confirmed the root cause and formation pattern of these impurities.
Methylphenidate and its related impurities were measured using various analytical techniques. Spectrophotometric, RP-HPLC and LCMS methods [
Methylphenidate working standard and impurity standards (
Water HPLC with 2695 separation module equipped with 2996 PDA detector used for the HPLC analysis. Acquity UPLC-MS/MS was used for the identification of the unknown impurities. The output signal was monitored and processed using empower 3 software. Cintex digital water bath was used for hydrolysis studies. Thermal stability studies were performed in a dry air oven (Cintex, Mumbai, India).
The chromatographic column used was a Symmetry Shield RP 18, 150 × 4.6 mm, 5.0 μm. The separations was achieved on a gradient method. Solvent A is a mixture of Buffer: Acetonitrile (92:8(V/V) [Buffer: 6.8 g of monobasic potassium phosphate and 2.0 g of Sodium 1-octane sulfonate monohydrate in 1000 mL water adjusted to pH 2.2 ± 0.05] and solvent B is mixture of Acetonitrile and THF in the ration (95:5). The flow rate was 1.5 mL∙min−1 and the detection wavelength was 265 nm. The HPLC gradient program was set as: Time (min)/% solution B: 00/15, 8/15, 40/40, 41/15, 50/15. The column temperature was maintained at 40˚C and the injection volume was 20 μL. 1% phosphoric acid was used as a Diluent.
LC-MS/MS system (Acquity UPLC coupled with TQD mass spectrometer with empower software, Waters Corporation, Milford, USA) was used for the identification of unknown compounds formed during forced degradation and stability testing studies. The method was developed using Acquity UPLC BEH C18 1.7 µm, 2.1 × 150 mm column as a stationary phase. The mobile phase A is a 0.1% of acetic acid in water. The mobile phase B is acetonitrile. The UPLC gradient program was set as: Time (min)/% solution B: 00/15,5.58/15,26.92/ 40,26.98/15,35.00/15. The column temperature was maintained at 40˚C and the injection volume was 15 μL. Milli-Q water was used as Diluent. The mobile phase pumped at 0.64 mL/min−1. The eluted compounds were monitored at 210 nm. The run time was 35 min. Mass spectrometric conditions optimized as cone gas 10 V, Collision flow 70 L/Hr., Ion energy 0.7 Entrance and Exit Potentials 1V, source temperature 150˚C, Desolova- tion gas 800 L/hr., Desolvation temperature 400˚C.
Analytical method developed and validated on HPLC which separates all the impurities (
Analytical methodology described in the section 2.3 was used for the analysis of compatibility studies. Compatibility studies were performed for all the excipients as per the composition of finished product. All the utilized individual excipients were taken as per the composition with drug and exposed to temperature at 80˚C for 16 Hours. After the specified time, volumetric flasks were withdrawn from hot air oven and allowed to reach room temperature. After reaching room temperature, samples were diluted with diluent up to 20 mL. All the samples were injected as per the method.
Stability sample analysis of methylphenidate oral solution was performed using method described in the section 2.3. Accelerated samples for Initial, 1, 2 and 3 months and controlled room temperature stability samples (initial, 3,6,9,12,18 and 24 months) were analyzed. The quantitative measurements of these esters formed in the finished product measured using validated analytical method. Due to the non-availability of the reference standards for the methylphenidate-glycerin esters, Quantitation was performed by using methylphenidate standard assuming that the same molar absorbance for both active and the formed impurities. For erythro isomer and ritalinic acid respective standards were used to quantify these impurities in the finished product.
Two lots of methylphenidate preparations were purchased. Both preparations are 5 mg/5 mL contains methyl phenidate, citric acid anhydrous, glycerin, N&A grape flavor, PEG 1450, and purified water. The expiry dates of these preparations were 11/2012, 5/2013.
To carry forward the structural elucidation of the unknown impurity Mass spectrometric analysis was performed. Identical UPLC method developed based on the HPLC of Methylphenidate related substance method due to the ion pair buffer in the HPLC method of analysis. Elution patterns were compared by developing an UPLC-MS/ MS method with volatile buffer and found sufficient separation of the impurity of interest.
The toxicology and mutagenicity endpoint of the formed Methylphenidate-glycerin esters were evaluated using the Lasha Sarah Nexus Trail software version 1.1.2 which will give the mutagenicity endpoint as per the ICH M7 guidelines.
There were several tailbacks in the process to identify the structure of the impurity. Analytical method was developed using ion pair buffer because of the hydrophilic nature of the Methylphenidate and also for the separation of 6 impurities from the main peak. In the reversed-phase chromatography nitrogen compounds were attached to chemically bonded silica which gives peak tailing, poor separation and reproducibility problems. These problems are probably due to the presence of residual silanol groups on the surface of the column material. 1-Hexane sodium sulfonate (anionic ion pairing agent) was added to the mobile phase. The ion-pair reagent is attracted to the stationary phase because of its hydrophobic alkyl group, and the charge carried by the reagent (C6-SO3−) there by attaches to the stationary phase. This negative charge on the stationary phase is balanced by positive ions (Na+) from the reagent. A positively charged sample ion (protonated base) can now exchange with Na+ ion, resulting in the retention of the sample ion by an ion exchange process. By this way hexane sulfonate adjusts the retention and improves the resolution.
In order to run the samples in LC-MS/MS method, it was decided to develop a method compatible for mass spectrometry. The ion pair buffer and phosphate buffer used in the HPLC method was not compatible with UPLC-MS/MS. So that UPLC method was developed using volatile buffer in the mobile phase. 0.1% formic acid in water was used as a mobile phase to match with the lower pH of HPLC buffer. Electrospray Ionization of positive ion mode was selected for the mass spectrometric analysis. 0.1% formic acid in water was used as a diluent instead of 0.1% phosphoric acid. Same Identical Pattern was reproduced using the above conditions. To identify the unknown impurities in the UPLC method, spectral characteristics were compared and observed that there is no significant change in the UV Spectra of impurities in consideration with other peaks. To confirm the unknown peaks of interest, elution pattern and % area of the unknown peaks in HPLC (
The unknown peaks were observed in the excipient compatibility studies, when glycerin was added to methyl- phenidate (
formation of the impurities is due to the Glycerin. Spectral characteristics of the impurity found in Methyl- phenidate oral solution at RRT 0.75 and 0.77 min was exactly matches with the impurity observed in the glycerin excipient compatibility experiment. Molecular ion [M+H] + shows intense ion of 294 confirms the addition of glycerin moiety with Methylphenidate (
Glycerin (
There were several articles described the formation of glycerin transesterification products in which glycerin is tend to form 1-mono and 2-mono esters when reacts with esters [
Forced degradation of the finished product shows the methylphenidate-glycerin esters in acid, base conditions. The formation of the ritalinic acid is the major impurity in all forced degradation conditions. About 1% Methylphenidate esters found in acid degradation@80˚C/2 Hrs. and ~2% of the esters found in Base degradation @ Room temperature/30 min. Based on the forced degradation results, methylphenidate degrades rapidly in Base hydrolysis. The formation of Erythro isomer is very minimal in all the stressed conditions. The chromatograms and results described in
Stress Condition | Reaction Time/Conditions | Purity Angle | Purity Threshold | % Deg |
---|---|---|---|---|
Control Sample-1 | NA | 0.055 | 0.290 | NA |
Control Sample-2 | 0.050 | 0.282 | ||
Acid Degradation | 5.0 mL of 1N HCl/80˚C/2 hour | 0.060 | 0.292 | 15 |
Base Degradation | 5.0 mL of 0.1N NaOH/RT/30 minutes | 0.057 | 0.281 | 33 |
Oxidation | 5.0 mL of 3%H2O2/80˚C/4 hours | 0.075 | 0.322 | 1 |
Thermal | Thermal/80˚C/4 hours (Use water bath) | 0.045 | 0.280 | 1 |
UV Light | UV Light/24 hours | 0.064 | 0.308 | 0 |
Spiked Sample | NA | 0.098 | 0.331 | NA |
Stress Condition | Name of the Peak | RT | RRT | % Area |
---|---|---|---|---|
Base Degradation | Glycerin Ester- 1 | 13.72 | 0.70 | 3.63 |
Glycerin Ester- 2 | 14.15 | 0.72 | 2.9 | |
Related Compound A | 14.66 | 0.75 | 20.4 | |
Erythroisomer | 15.77 | 0.81 | 1.42 | |
Acid Degradation | Glycerin Ester- 1 | 13.68 | 0.70 | 1.00 |
Glycerin Ester- 2 | 14.11 | 0.72 | 0.78 | |
Related Compound A | 14.62 | 0.75 | 12.36 | |
Erythroisomer | 15.72 | 0.81 | 0.34 | |
Peroxide Degradation | Related Compound A | 14.7 | 0.75 | 0.14 |
Erythroisomer | 15.75 | 0.81 | 0.02 | |
Thermal Degradation | Related Compound A | 14.53 | 0.75 | 0.18 |
40˚C /75%RH Stability Summary | ||||
---|---|---|---|---|
Condition | Ritalinic acid | Erythro isomer | Ester-1 (RRT 0.75) | Ester-2 (RRT 0.77) |
Initial | 0.00 | 0.00 | 0.00 | 0.01 |
1 Month | 0.40 | 0.00 | 0.055 | 0.06 |
2 Months | 0.797 | 0.092 | 0.106 | 0.118 |
3 Months | 1.301 | 0.136 | 0.164 | 0.162 |
25˚C/60%RH Stability Summary | ||||
Initial | 0.00 | 0.00 | 0.00 | 0.00 |
3 Months | 0.277 | 0.00 | 0.00 | 0.00 |
6 Months | 0.562 | 0.00 | 0.101 | 0.081 |
9 Months | 0.768 | 0.098 | 0.142 | 0.087 |
12 Months | 1.102 | 0.112 | 0.168 | 0.16 |
18 Months | 1.444 | 0.125 | 0.241 | 0.198 |
24 Months | 2.061 | 0.172 | 0.348 | 0.318 |
Quantitative analysis of finished product was performed in CRT (Controlled Room Temperature) and accelerated conditions. The formation of the impurity is linear and doesn’t degrades after the formation. The results were extrapolated in the graph described in
Two lots of commercial liquid preparations of methylphenidate with different expiry dates were analyzed (
The formed products of these methylphenidate-glycerin ester structures were evaluated using lasha Sarah nexus trail software for the mutagenicity. The Mutagenicity endpoint of these two compounds (
It has been shown that the drug substance methylphenidate reacts with glycerin to form esters. Results of the stability samples, forced degradation studies and excipient compatibility studies confirmed these esters. The formed esters are above the identification limits based on the maximum daily dose of the methylphenidate. Mutagenicity of these esters found negative up to 75%. Commercially available marketed preparations confirm the formation of these impurities 0.6%.
The authors wish to thank the management of Novel Laboratories INC for providing the infrastructure for the supporting of this research work. Cooperation from colleagues Quality Control and Analytical Research& Development of Novel Laboratories is appreciated.
Kishore KumarHotha,SwapanRoychowdhury,VeerappanSubramanian, (2016) Drug-Excipient Interaction of Methylphenidate with Glycerin in Methylphenidate Oral Solution and Identification of its Transesterification Products by UPLC-MS/MS. American Journal of Analytical Chemistry,07,151-164. doi: 10.4236/ajac.2016.72013