The Application Of HPLC-MS Technology In The Characterization Of Degradation Products And Impurities In Drug

The quality of drug has a close link to human health and thecharacterization of degradation products and trace impurities has also beenpaid more and more attention. Control of impurities has been an important partof the quality assurance of drug for the existence of impurities can result in aloss of drug activity or affect the stability of the drug, even initiate possibleside effects and adverse reactions in clinical medicine. The presence ofimpurities in drug products may be harmful to human health. Understandingthe formation of degradation products is very important to predict drugstability problems, define product manufacturing conditions and choosesuitable packaging and storage conditions. The result may also be useful toestablish the degradation pathway of the drug and verify the feasibility of theanalytical procedure.Lacosamide is a new type of antiepileptic drug, which has been used asadd-on therapy for partial-onset seizures in adults. It reveals that only oneliterature of lacosamide has been reported and some degradation productswere still unidentified. Ampicillin belongs to semi-synthetic β-lactamantibiotic which has been widely used for the treatment of infections. However,it was found that some degradation products of ampicillin sodium could notyet be identified with available reference substances or literatures. Thus, inorder to better control the quality of these drugs, it was necessary to establishan effective method for the rapid characterization of unknown degradationproducts and impurities.High performance liquid chromatography-mass spectrometry (HPLC-MS)technology has been a powerful tool for the impurity analysis of drug, whichpossesses the capabilities of high separation and powerful structuralelucidation. In the present study, HPLC-MS technology was performed to investigate the impurities and degradation products in lacosamide andampicillin sodium. Forced degradation characteristics of lacosamide andampicillin sodium under various stress conditions were studied anddegradation products were elucidated and characterized. These results wereexpected to provide new information for the quality control and identificationof impurities in drugs.Part one The characterization of degradation products from lacosamideby HPLC-MS/MSObjective: To characterize degradation products of lacosamide underacidic, alkaline and oxidative stress conditions with the combined use of highperformance liquid chromatography hybrid triple quadrupole-linear ion trapmass spectrometry (HPLC-QTrap-MS) method and high performance liquidchromatography hybrid ion trap/time-of-flight mass spectrometry(HPLC-IT-TOF-MS) method.Methods: A HPLC-QTrap-MS method was performed for theidentification and characterization of degradation products from lacosamideunder acidic (1mol/L HCl), alkaline (1mol/L NaOH) and oxidative (30%H2O2) stress conditions. Possible molecular masses and structures wereproposed based on the information of MS spectra. Accurate masses andelemental compositions of degradation products and product ions weredetermined by HPLC-IT-TOF-MS, which could also support the previousinference results. The chromatographic separation was performed on anAgilent Zorbax SB-C18(150mm×4.6mm,5μm) column, with ammoniumacetate (2mmol/L,0.1%formic acid) and acetonitrile as gradient eluents. Themobile phase flow rate was set at0.8mL/min.Results: A total of7degradation products of lacosamide have beenanalyzed, of which4products were identified and3others were partiallycharacterized. The four degradation products were separately identified as(R)-2-amino-N-benzyl-3-methoxypropanamide (m/z209),2-acetamido-N-benzylacrylamide (m/z219),2-acetamido-N-(hydroxy (phenyl)methyl)-3-methoxypropanamide (m/z267) and 2-acetamido-N-benzyl-3-hydroxypropanamide (m/z237).Conclusions:1The method was sensitive and effective to separatelacosamide from its degradation products. Mass information was rich andaccurate for the characterization of degradation products.2Degradationproducts of lacosamide were different under acidic, alkaline and oxidativestress conditions, of which (R)-2-amino-N-benzyl-3-methoxypropanamide(m/z209) was the main degradation product.3Compounds, including2-acetamido-N-benzylacrylamide (m/z219),2-acetamido-N-(hydroxy (phenyl)methyl)-3-methoxypropanamide (m/z267) and2-acetamido-N-benzyl-3-hydroxypropanamide (m/z237), were first reported.Part two The characterization of degradation products from ampicillinsodium by HPLC-MS/MSObjective: To study the degradation characteristics of ampicillin sodiumand characterize its degradation products under various stress conditions withthe combined use of HPLC-QTrap-MS and HPLC-Q-TOF-MS (highperformance liquid chromatography hybrid triple quadrupole-time offlight-mass spectrometry).Methods: Ampicillin sodium was subjected to forced degradation studiesunder acidic, alkaline, hot, photolytic and humid stress conditions. Thecombined use of HPLC-QTrap-MS scanning modes consisting of enhancedmass spectrum-enhanced resolution-information dependentacquisition-enhanced product ion (EMS-ER-IDA-EPI), precursorscan-enhanced resolution-information dependent acquisition-enhancedproduct ion (PREC-ER-IDA-EPI) and enhanced product ion (EPI) wasperformed on the degradation products for structural elucidation. Accuratemasses and elemental compositions of degradation products and product ionswere obtained by using HPLC-Q-TOF-MS, which could also be used to verifythe inference results. The chromatographic separation was performed on anAgilent Zorbax SB-C18(150mm×4.6mm,5μm) column, with ammoniumacetate (1mmol/L) and acetonitrile as gradient eluents. The mobile phase flowrate was set at0.8mL/min. Results: A total of19degradation products and impurities under all theabove forced conditions were identified and characterized,4products ofwhich have not been reported. These degradation products were separatelyidentified as(Z)-2-oxo-2-(((2-oxo-3-phenyl-2,3,6,7-tetrahydro-1H-1,4-diazepin-5-yl)methylene)amino)-1-phenylethanaminium (m/z349),(E)-2-(((2-(2-amino-2-phenylacetamido)-2-phenylacetyl)imino)(carboxy)methyl)-5,5-dimethylthiazolidine-4-carboxylic acid (m/z499),(E)-1-carboxy-N-((3,6-dioxo-5-phenyl-1,6-dihydropyrazin-2(3H)-ylidene)methyl)-2-mercapto-2-methylpropan-1-aminium (m/z348) and5-(amino(phenyl)methyl)-7-formyl-2,2-dimethyl-2,3-dihydroimidazo[5,1-b]thiazole-3-carboxylic acid (m/z332).Conclusions:1HPLC-Q-TOF-MS method was sensitive, specific andaccurate, which could provide more structural information. In the presentstudy, the developed method could be effectively used to separate ampicillinfrom its degradation products and characterize its degradation products.2Thecombined use of EMS-ER-IDA-EPI, PREC-ER-IDA-EPI and EPI scan modeshas been effectively used for the characterization of unknown impurities anddegradation products in drugs.Part three Analysis of impurities in ampicillin sodium by HPLC-MS/MSObjective: To develop a sensitive and effective HPLC-QTrap-MSmethod for the identification of impurities in ampicillin sodium and comparethe quality of ampicillin sodium with its foreign commercial sample.Methods: The enhanced mass spectrum (EMS) scan mode of HPLC-QTrap-MS was performed for the analysis of ampicillin sodium and its foreigncommercial sample.1Ampicillin for system suitability (including ampicillin,ampicilloic acid, diketopiperazines of ampicillin, open-cycle ampicillin dimer,closed ampicillin dimer and open-cycle ampicillin trimer) was used to identifythe known impurities by comparing the retention times and mass spectra.2For the identification of unknown impurities, molecular mass could bededuced by the ions of [M+H]+,[M+Na]+and [M+K]+. Structural elucidation of unknown impurities could be performed using the product ions provided bythe EPI mass spectra. The chromatographic separation was performed on aKromasil C18(150mm×4.6mm,5μm) column, with acetic acid (pH3.4) andmethanol as gradient eluents. The mobile phase flow rate was set at0.8mL/min.Results:1Impurities, including ampicilloic acid, diketopiperazines ofampicillin, open-cycle ampicillin dimer and closed ampicillin dimer, could bedetected both in ampicillin sodium and its foreign commercial sample.Open-cycle ampicillin trimer has only been detected in foreign commercialsample.2Three other degradation products were separately proposed to beL-ampicillin, D-phenylglycylampicillin and closed ampicillin trimer accordingto the information of mass spectra.Conclusions:1Compared to the foreign commercial ampicillin sodium,fewer impurities have been detected in ampicillin sodium, which couldprovide the basis for the quality control of ampicillin sodium.2The methodwas sensitive and effective for the separation of ampicillin from its impurities.Unknown impurities could be characterized based on the mass information.