Developing a Bioanalytical Method for an Immunomodulator Compound
Challenge: A sensitive and specific bioanalytical method suitable for quantification of an immunomodulator compound in plasma matrix for human, rat and mouse samples at a low detection level, LLOQ = 10 pg/mL, was developed by Diteba's scientists for pre-clinical and clinical studies. This compound is a member of a class of immunomodulatory drugs that possess γ-glutamyl or β-aspartyl moieties, which was discovered by Russian scientists and is being examined for efficacy in several indications. The synthesized molecule contains two chiral centers and therefore can exist as four configurations: two enantiomers and two diastereoisomers.
Solution: The advancement of this research was due to the selection of an unconventional chiral mobile phase technique for the separation of enantiomers and diastereoisomers. This technique has several advantages compared to chiral stationary phase. For instance, it employs a chiral stationary phase, which is less expensive and more rugged than a chiral stationary phase. At the same time, it allows more flexibility because a column can be used in conjunction with several additives simultaneously or chiral additives can be eluted out of the column with strong solvent and a subsequent additive can be used. A further advancement achieved by Diteba in this research was the facile derivatization procedure coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace level of the compound in biological samples.
In order to develop an assay method, Diteba's scientists had to ensure that the analyte molecule having the DD configuration was well separated from the other isomers (LL, LD and DL) that might be present in plasma samples as endogenous compounds. The presence of either enantiomer (LL) or poor resolved diastereomers (DL or LD) as endogenous background in the blank plasma samples will interfere and make it impossible for the accurate quantification of low dose active in study samples.
The UV detection method showed a lack of sensitivity; therefore, a more sensitive fluorescence detection technique was developed. A 3D scan was used to select appropriate Excitation and Emission wavelengths. It was found that the detection limit (DL) for LL and DL isomers was 500 pg/mL and for DL and DD 1 ng/mL. Three different lots of human plasma were screened in order to identify contents of suspected endogenous analytes: LL and DL; DD and LD were optional for this study. In order to confirm the correct assignment of peaks and to obtain chromatographic reproducibility, the sample preparation (extraction) procedure was replicated for both human and animal plasma.
The general approach for confirming the identity of targeted analytes was to spike plasma samples, before and after extraction, with neat solutions containing corresponding LL, DL (or DD, LD) and compare them with the marker solution. These neat, LL, DL and DD, LD solutions were prepared at the same time and ran in the same UPLC sequence as the plasma samples.
Result: Diteba achieved highly efficient separation of all four isomers that allowed screening of both the human and animal plasma for contents of endogenous compounds; however still, the method was not able to achieve quantification of the active at the requested low level (10 pg/mL). Therefore, chemical derivatization was necessary to increase detection sensitivity. For this purpose, a number of different types of tagging reagents were evaluated. A novel synthesized fluorescent reagent, BCEOC was found to be suitable for the proposed assay method. Identification of derivatives was accomplished by liquid chromatography/electrospray ionization mass spectrometry UPLC/MS/MS. The derivatives exhibited strong fluorescence; the extracted derivatization solution demonstrated excellent yields and the detection limits were in the femtomole range.
Diteba also developed and verified two unique analytical methods for biological sample testing. A selective and sensitive chiral UPLC method for the separation, identification and quantification of this immunomodulator compound and for screening contents of endogenous compounds in different biological matrixes was developed and successfully applied. This method is based on fluorometric detection that monitors native florescent moieties of the molecules. It was found that the dominant form in screening species is the DL isomer and not LL which previously had been suspected when other two isomers LL, LD are presented at trace levels. The effects of different parameters such as mobile phase composition, temperature, chiral additive reagent and the structure of the analytes on the selectivity in both chromatographic modes were investigated. By varying the parameters, the separation of the stereoisomers was optimized and, as a result, baseline resolution was achieved.
A pre-column derivatization method for the determination of the compound using the tagging reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by UPLC with fluorescence and MS/MS detection was also developed by Diteba. BCEOC can easily and quickly label either peptides or amino acid molecules in the analyte. After derivatization, four diastereoisomers DD-L-tag, LL-L-tag, DL-L-tag and LD-L-tag were separated and identified using a reverse phase UPLC column (BEH C18, 2.5 x 150mm, 1.7μm) by fluorescence detection and mass spectrometry (UPLC/MS/MS). Derivatives are stable enough to be efficiently analyzed by Ultra Performance Liquid Chromatography. The detection limit for the analyte was calculated at 1.0 pmol on column in a 10 μL injection volume with a signal-to-noise ratio of 3. The mean interlay precision for all standards was <10% of the expected concentration. Excellent linear responses were observed with a coefficient of 0.9999. Therefore, the facile derivatization procedure coupled with mass spectrometry allowed the development of a highly sensitive and specific method. To Diteba's knowledge, this was the first successfully implemented method for the quantitative analysis of trace level of this immunomodulator compound in biological samples.