Richard King (Presenter)
PharmaCadence Analytical Services
Authorship: Richard King(1), Susan Crathern(1), Robyn Buerger(1), Lorin Bachmann(2)
1) PharmaCadence Analytical Services, Hatfield, PA 2) VCU Health System
| Short Abstract Interference testing is a critical component of the method validation process for a routine clinical laboratory test. One source of potential interferences in LC-MS/MS are the phase II metabolites of the analyte. While it is possible to purchase some of the more common glucuronide metabolites for testing, they are expensive and not all are stable. By using human liver microsomes fortified with the appropriate co-factors, the glucuronide conjugates can be produced in sufficient quantities to perform interference testing at a relatively low cost. Results will be shown for steroid hormones Androstenedione, Desoxycortisol, Cortisol, DHEA, Deoxycorticosterone, 17-Hydroxypregnenolone, Progesterone, 17-Hydroxyprogesterone, and Testosterone. |
Long Abstract
Interference testing is a critical component of the method validation process for a routine clinical laboratory test. Interference testing for LC-MS/MS should include assessment of molecules with the potential to produce similar precursor and product ions compared to the analyte. One such source of potential interferences in LC-MS/MS are the phase II metabolites of the analyte itself. One of the most common phase II metabolites is the conjugate with glucuronic acid. These are typically products of the uridine 5'-diphospho-glucuronosyltransferase (UGT) family of enzymes and for many compounds, including steroids, can be an important metabolic pathway.
The addition of glucuronic acid to the parent molecule results in a mass addition of 176 Daltons. Ordinarily, this would be more than enough of a mass difference to preclude interference in the mass measurement. However, the bond formed is labile and can easily dissociate to yield the parent molecule as one of the products. Dissociation can take place in the ion source with heat and it may be the result of collision induced dissociation on the way into the high vacuum region of the mass spectrometer. If sample preparation and chromatography are not appropriate, the conjugates and the analyte will co-elute leading to artificially high results.
While it is possible to purchase some of the more common glucuronide metabolites for testing, they are expensive and not all are stable. By using in vitro systems fortified with the appropriate co-factors, the glucuronide conjugates can be produced in sufficient quantities to perform interference testing at relatively low cost. The methods and procedures described for making the glucuronides are common practice in pharmaceutical research. One such method using human liver microsomes for production of glucuronide conjugates will be described and results will be shown for steroid hormones Androstenedione, Desoxycortisol, Cortisol, DHEA, Deoxycorticosterone, 17-Hydroxypregnenolone, Progesterone, 17-Hydroxyprogesterone, and Testosterone.
Human liver microsomes at 0.5 mg/ml total microsomal protein were incubated at 37 C for two hours in the presence of 10 ug/ml steroid substrate, 1 mM uridine 5'-diphospho-glucuronic acid (UDPGA), and 25 ug/ml alamethicin. Reactions were initiated with the addition of UDPGA and quenched by the addition of two volumes of acetonitrile containing 0.1% formic acid. The samples were centrifuged to pellet the remaining protein, the supernatant removed to a clean injection plate and diluted two fold with water. All analyses were conducted in positive ion mode using a 1.0 mm by 50 mm Luna C18 column from Phenomenex. The LC separation was carried out using an Eksigent Micro LC200 from Sciex. The formation of glucuronide was confirmed by running a product ion scan of the predicted precursor m/z for the glucuronide.
If the glucuronide is separated chromatographically from the analyte then there is no interference concern. If conjugates are found to interfere, then the sample preparation procedures or chromatography conditions must be altered to remove the conjugate interference.
The presentation will include complete instructions for performing the microsomal incubations, determining the expected glucuronide conjugate m/z, and building the predicted glucuronide SRMs. Conjugate interference data for the steroid hormones listed above will be shown.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | PharmaCadence Analytical |
| Board Member | no | |
| Stock | yes | PharmaCadence Analytical |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |