Ioning out low level impurities

Drug production requires high standards of purity for intermediates. Jerry Zweigenbaum explains how Agilent's LC/MSD Ion Trap can be used to detect impurities in the intermediates used

The production of fine chemicals requires high standards of purity because impurities can cause unwanted and deleterious reactions and by-products.If carried through to the final product, some impurities can be physiologically harmful, while others may be innocuous, having only little effect on the efficacy and concentration of the resulting drug.

Regardless of any effects, it is necessary to determine the identity of impurities and establish a tolerance level. This level could be set as some percentage of the 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 such extensive measures 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 the detection of impurities at low levels. Typically, the compound is dissolved at a high concentration and analysed 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 the LC/MSD Ion Trap. Sensitivity and the Auto MS/MS capability with the application of the powerful 'SmartFrag' are demonstrated.¹

The system used was an Agilent LC/MSD Ion Trap SL with an 1100 binary pump and well-plate autosampler. A StableBond C18 column 2.1 x 50cm 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 10mg/ml. Injections were made at 1ml. The ion trap was operated with SmartFrag setting from 20% to 200% of the fragmentation amplitude (set at 1.0 V).

precursor ions

The capillary exit and skimmer was set at a low energy to minimise 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 intermediate was necessary to detect the protonated molecule (in positive ion mode) of the impurities present and obtain MS/MS fragmentation of that precursor ion.

Example 1 involves a 1,4- Benzodiazepine intermediate. The compound 2-amino-5-chloro -2' -fluorobenzophenone (I) was analysed as an intermediate to the drug flurazepam (IV). Scheme I shows the reaction of this compound to form the drug.²

The total ion MS/MS chromatogram of the intermediate is shown in figure 1. An impurity is observed at a low level compared with the response of the intermediate. The LC/MSD Ion Trap detects the major ion in the impurity and performs an auto MS/MS, ramping the fragmentation amplitude from 0.2 to 2.0 V (Smartfrag). The resulting spectrum 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 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.

reaction pathway

This is consistent with fragmentation on both sides of the carbonyl bond. The proposed structure favours 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 50mm column, the chromatographic resolution is obtained.

Example 2 involves the ACE inhibitor Enalapril prescribed for chronic heart failure. A reaction pathway for the production of Enalapril is shown in Scheme II.

In this reaction the intermediate, N-(1-ethoxycarbonyl-3-phenyl propyl) alanine (I) is reacted with 1,1'-carbonyldiimidazole (CDI) and then further reacted with sodium proline to produce Enalapril (III).³ A concentrated solution of the intermediate analysed with the LC/MSD Ion Trap again, in figure 3, shows impurities. The auto MS/MS spectrum of the early eluting impurity and a proposed structure is given in figure 4. This impurity displays 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.

good separation

The MS/MS of the intermediate (not shown) gives 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 separation by LC and the power of auto MS/MS and SmartFrag demonstrates the ability to detect and identify impurities.

Example 3 is the intermediate (I) for flunarizine (III), 1-[(4-fluorophenyl) (phenyl)methyl]piperazine (I). A synthetic pathway for flunarizine, a calcium blocker used for migraine headaches, Scheme III

In this example Auto MS/MS was not used. The total ion single MS chromatogram of the intermediate is given in figure 5. 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 piperazine.

The addition of an 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.

These examples 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 separation and signal of the impurities detected. The 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 chromatographic retention time with an authenticated standard provides additional confirmation.