Determination of enantiomeric purity is of paramount importance, as the example of thalidomide has taught us that optical antipodes of a pharmaceutically active substance could exhibit devastating side effects.
For the determination of enantiomeric purity, we offer three different approaches: Detection and quantification by GC-FID, GC-MS or HPLC. Please explore advantages and drawbacks for each method in the following.
One of the most accurate and sensitive methods for the analysis of enantiomeric purity of free amino acids involves derivatisation and gas chromatographic separation of the enantiomers using chiral stationary phases. The flame ionization detector (FID) offers an extended linear range for an accurate quantification across a broad concentration range.
Besides all proteinogenic amino acids, this method also allows for the detection of almost all non-proteinogenic amino acids and most N-methyl-amino acids. Amino acids with more than one chiral centre (e.g. Isoleucine) are detected as four separate stereo isomers.
Depending on the sample, the preparation comprises the cleavage of resin bound amino acids and cleavage of protecting groups (e.g. Fmoc) and hydrolysis of protection groups. The resulting free amino acids are then derivatised in a two-step process. Basically, acidic groups are converted into esters, whereas free nucleophilic groups (e.g. amines, alcohols, thiols) are acylated. These steps increase the analyte’s volatility and render them susceptible to separation by gas chromatography. A third derivatisation step could be necessary for amino acids which contain additional functional groups (e.g. histidine).
Please note: harsh conditions during sample preparation (e.g. hydrolysis) could lead to amino acid racemization which results in an overestimation of antipode quantity. The overestimation is neglectable for samples where the minor enantiomer is present in larger quantities (1-5% or more, depending on the amino acid). However, the effect
becomes critical in cases where only small amounts of antipode (< 1% up to < 10% for cysteine) are present. Here, even small quantities of racemization due to sample preparation have a great impact on the already small amounts of natively present antipode. In latter cases, the determination of enantiomeric purity by GC-MS should be considered.
Our go-to protocol for a reliable quantification of minute amounts of enantiomeric antipodes, even for samples which were prepared under harsh conditions (e.g. hydrolysis, protecting group cleavage etc.). The GC-MS protocol follows the same sample preparation as our GC-FID protocol. However, a stringent regime of deuterated environment during sample preparation coupled with a mass selective detector allows for a reliable identification and quantification of optical antipodes even in high background scenarios such as peptide hydrolysates or even formulated samples.
Depending on the sample, the preparation comprises the cleavage of resin bound amino acids and peptides, cleavage of protecting groups (e.g. Fmoc) and hydrolysis of polypeptides. The resulting free amino acids are then derivatised in a two-step process. Basically, acidic groups are converted into esters, whereas free nucleophilic groups (e.g. amines, alcohols, thiols) are acylated. These steps increase the analyte’s volatility and make them susceptible to separation by gas chromatography. A third derivatisation step could be necessary for amino acids which contain additional functional groups (e.g. histidine).
Hydrolysis and each derivatisation step is performed in a deuterated environment. This means, that racemization which occurs as an artefact of hydrolysis and sample treatment is associated with the incorporation of a deuterium instead of a proton. Thus, the amounts of artificially introduced amounts of antipode can be distinguished from those antipodes which were natively already present in the sample prior to sample treatment.
This distinction is ensured, as the chromatographically separated analyte mixture is further analysed by a mass selective detector (MSD) in selective ion monitoring (SIM) mode. Here, the MSD only „looks“ at those analyte ions which are protonated – those natively present in the original sample. Deuterated counterparts are excluded by selection of appropriate SIM parameters.
Certain sample matrices could lead to an overestimation of the minor enantiomeric antipode. These matrices are commonly found in formulated samples and contain high concentrations of e.g. mannitol or buffers. These excipients need to be removed prior to sample hydrolysis and/or derivatisation as they could act as a proton donor and thereby disturb the deuterated environment necessary for antipode quantification.
The removal of these substance is usually performed by solid phase extraction (SPE) on C18 reversed phase materials or by ion exchange. A prerequisite to this method is a pronounced polarity difference between analyte and to-be-removed excipients. A rather hydrophobic analyte is captured on the SPE material, whereas the highly water soluble excipients are flushed through the SPE material without retention. The analyte is then selectively eluted from the SPE material and subjected to further treatment.
We offer a modified version of our A.0.3. protocol for the quantification of free amino acids and amino acid derivatives which require only short hydrolysis times. Peptides, even as short as a dipeptide or pseudoproline building block are not suited for this modified method.
This protocol is limited to aforementioned cases, but offers the advantage of improved specifications compared to A.0.3.: Range: 0.05 to 1.5%; LOD: <<0.1%; LOQ; 0.05%; SD: ≤± 0.03% at ≤ 1.5% Enantiomer. The analysis is further validated by the initial analysis of a performance qualification (PQ). Here, a sample of known antipode content is analysed and must satisfy acceptance criteria prior to analysis of the customer’s sample.
Determination of enantiomeric purity by HPLC offers advantages through a straightforward sample preparation when compared to the multistep derivatisation approach for determination by GC. The analyte is detected and quantified in its native form. The absence of a complex sample preparation alleviates artificial racemazation due to harsh conditions during sample preparation. The prerequisite for this method is the presence of a chromophoric group such as the Fmoc protecting group. This allows for the analyte detection and quantification by its characteristic UV spectrum.
The native analyte is subjected to chromatographic separation on a chiral stationary HPLC phase. Eluting analytes are detected by diode array detector (DAD) and quantified based on the integration of their UV spectrum at a specific wavelength.