Agilent Fine Chemicals: Identification of Impurities in Drug Intermediates by LC/MSD Ion Trap Application Note
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1. Fine Chemicals Identification of Impurities in Drug Intermediates by LC MSD lon Trap i 2 e Application e e 8 o e ee a e 8 e Specialty Chemical e Authors J Zweigenbaum Agilent Technologies Inc 2850 Centerville Road Wilmington DE 19808 1610 USA Abstract The production of fine chemicals requires high standards of purity for drug intermediates Using the Agilent LC MSD lon Trap sensitive impurity detection was demonstrated for intermediates used in the production of the drugs flurazepam enalapril and flunarizine For each intermediate LC MS and MS MS data were obtained in one run and the results provided sufficient information to propose structures for the corresponding impurities Introduction Production of fine chemicals requires high stan dards of purity for drug intermediates and other critical applications Impurities can cause unwanted and deleterious reactions and by products If carried through to final product some impurities can be physiologically harmful Others may be innocuous having little effect on efficacy and concentration of the resulting drug Regardless it is necessary to determine the identity of impurities and establish a tolerance level This level could be set as some per centage of total signal measured Authenticated standards of the impurity may need to be prepared to confirm the identity and provide a quantitative measure The need for extensive measure2. ation and signal of the impurities detected The resulting data provided enough information to propose structures It must be noted that MS MS alone is insufficient for positive identification of an unknown Comparison of the MS MS spectrum with an authenticated reference is required Information such as matching chro matographic retention time with an authenticated standard provides additional confirmation References 1 Paul Goodley Maximizing MS MS Fragmenta tion in the Ion Trap Using CID Voltage Ramping Agilent Technologies publication 5988 0704EN www agilent com chem 2 Norman W Gilman et al 1990 Atropisomers of 1 4 Benzodiazepines Synthesis and Resolu tion of a Diazepam Related 1 4 Benzodiazapine Journal of the American Chemical Society 112 3969 3978 For More Information For more information on our products and services visit our web site at www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequen tial damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2010 Printed in the USA October 7 2010 5988 8236EN a Agilent Technologies
3. diate was necessary to detect the protonated molecule Cin positive ion mode of impurities present and obtain MS MS fragmentation of that precursor ion a Agilent Technologies Results Example 1 1 4 Benzodiazepine intermediate The compound 2 amino 5 chloro 2 fluoroben zophenone 1 was analyzed as an intermediate to the drug flurazepam IV Scheme I shows the reac tion of this compound to form the drug 2 The total ion MS MS chromatogram of the interme diate is shown in Figure 1 An impurity is observed at a low level as compared to the response of the intermediate The LC MSD Ion Trap detects the major ion in the impurity and performs an auto MS MS ramping of the fragmentation amplitude from 0 2 to 2 0 V SmartFrag The resulting spec trum provides sufficient information to propose a structure The single MS data shows that the M H ion is the same as the intermediate It also Scheme a HN i F N o D S CH3 HN 0 F l I H3C cl Il N H3C 0 N A H3C CH3 N CHCl F NaN3 i 0 ral _ gt l Vi m mn IV Flurazepam gives an isotope ratio consistent with one chlorine as with the intermediate However the MS MS fragmentation of the impurity shows only a major product ion at m z 123 The intermediate s MS MS produces an m z 154 and a less intense m z 123 product ion This is consistent with fragmentation on both sides of the carbonyl bond The proposed structure favors
4. luting impurity and a proposed structure is given in Figure 4 This impurity dis plays a single MS spectrum with an ion at m z 266 This is 14 amu less than the intermediate and suggests the impurity contains one less methylene The MS MS of the intermediate not shown gives II prominent m z 234 and 206 product ions Loss of formaldehyde would produce the m z 234 Loss of ethylformate produces the m z 206 In the impurity MS MS spectrum the m z 206 is present suggesting preservation of the phenylpropylalanine The m z 220 suggests the methylene is missing from the ester Again the combination of good separa tion by LC and the power of auto MS MS and SmartFrag demonstrates the ability to detect and identify impurities Impurity Intensity x 108 0 0 T T T T T T 0 2 4 6 8 10 Time min Figure 3 Total ion chromatogram of auto MS MS showing impurity separated from intermediate of Enalapril 4 206 1 Intensity x 104 N f m rri rrr ir ril Tri rri a TIT rii 50 100 150 200 250 300 350 400 450 500 m z Figure 4 Auto MS MS of impurity in N 1 ethoxycarbonyl 3 phenyl propyl alanine with proposed structure Example 3 A third example is the intermediate 1 for flunar izine IID 1 4 fluorophenyl phenyl methy piper azine 1 A synthetic pathway for flunarizine a calcium blocker used for migraine headaches is shown in Scheme III In this example Auto MS MS was not used The total i
5. on single MS chro matogram of the intermediate is given in Figure 5 Scheme Ill a oO Y l O N He A ra HO I Q g 1 m Flunarizine Impurity Intensity x 106 T T T T T T T T Tod 1 2 3 4 5 6 7 8 9 Time min Figure 5 Total single MS ion chromatogram of flunarizine intermediate The arrow indicates the impurity with m z 317 A second analysis was performed using MS MS of an impurity peak with m z 317 and the resulting spectrum is shown in Figure 6 with a proposed structure Both the intermediate and the impurity show a prominent m z 203 suggesting the loss of the piperazine moeity The addition of the ethyl group would explain the data In this analysis a minimum of two experiments one MS and one MS MS were required without auto MS MS to attain sufficient information to propose a structure m C Intensity x 105 m z Figure 6 MS MS of m z 317 with proposed structure in the analysis of the intermediate 1 4 fluorophenyl phenyl methyl piperazine www agilent com chem Conclusions Three examples of fine chemicals have been used to demonstrate the power of the LC MSD Ion Trap for identification of impurities Agilent s SmartFrag and Auto MS MS provided the capability to obtain precursor ion and representative fragmentation in one LC MS and MS MS run The robustness of the orthogonal sprayer and Agilent StableBond column allowed overload of the subject chemical with good separ
6. s as these would wholly depend on the identification and evaluation of detected compounds Liquid chromatography ion trap mass spectrometry the Agilent LC MSD Ion Trap is ideally suited for the screening of intermediates for detection of impurities at low levels Typically the compound is dissolved at a high concentration and analyzed for impurities The chromatographic method needs to provide sufficient separation of the material from its impurities This article provides examples that show the unique and powerful capabilities of Agilent s LC MSD Ion Trap Sensitivity and the Auto MS MS capability with the application of the powerful SmartFrag are demonstrated 1 Experimental The system used was an Agilent LC MSD Ion Trap SL with an 1100 binary pump and well plate autosampler A StableBond C s column 2 1 x 50 mm with 10 mM ammonium acetate and acetonitrile as the mobile phase was used A gradient from 10 to 90 organic produced separation and elution of all compounds present Intermediates were prepared at concentrations of 5 to 10 mg mL Injections were made at 1 uL The ion trap was operated with SmartFrag setting from 20 to 200 of the frag mentation amplitude set at 1 0 V The capillary exit and skimmer was set at a low energy to mini mize fragmentation in the ion transport region This was done using Smart Parameter Settings with a compound stability of 20 With auto MS MS turned on only one run per interme
7. the cleavage of the amido bond and neutral loss of chloroaniline The spectrum and the proposed impurity are shown in Figure 2 The StableBond column shows excellent stability at the low pH of the chromatographic conditions and even with a 2 1 x 50 mm column the needed chromatographic resolution is obtained J Impurity 1 5 4 2 4 F J E o et 05 Wenner o Life 0 0 0 2 4 6 8 10 12 Time min Figure 1 Total ion chromatogram auto MS MS of flurazepam intermediate 2 amino 5 chloro 2 fluorobenzophenone Note the good separation obtained for the impurity denoted by the arrow using the StableBond C4 2 1 x 50 mm rapid resolution column 4 123 0 x E SSS 50 100 150 200 250 350 400 450 450 m z Figure 2 Auto MS MS of impurity in 2 amino 5 chloro 2 fluorobenzophenone with proposed identity Example 2 Another example involves the ACE inhibitor Enalapril IlI prescribed for chronic heart failure A reaction pathway for the production of Enalapril is shown in Scheme II Scheme Il 1 col NH 0 i HC OH CH3 III 0 0 HC In this reaction the intermediate N 1 ethoxycar bonyl 3 phenyl propyl alanine I is reacted with 1 1 carbonydiimidazole CDI and then further reacted with sodium proline to produce Enalapril II A concentrated solution of the intermediate analyzed with the LC MSD Ion Trap again in Figure 3 shows impurities The auto MS MS spec trum of the early e
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Agilent Fine Chemicals: Identification of Impurities in Drug Intermediates by LC/MSD Ion Trap Applic