Ravi Orugunty (Presenter)
Cerilliant
Bio: Personal Biographical Sketch: Ravi Orugunty has been in the field of Bioanalytical chemistry for 10 years. He is currently a Senior Scientist at Cerilliant (a Sigma Aldrich company). He is involved in the Research and Development of LCMS Methods and Validation of Bioanalytical Methods. He is also involved in Development of Matrix based calibrator products at Cerilliant.
Authorship: Ravi Orugunty2, Uma Sreenivasan2, Josh Cooper2, Isil Dilek2, Linda Nagore2 Jackie Day1, James J Walters1 and Kevin Ray1
1 – Sigma-Aldrich Corporation, Applied Research and Development, 2909 Laclede Ave., St. Louis MO; 2 – Cerilliant Corporation, 811 Paloma Dr., Round Rock, TX
| Short Abstract Development of accuracy based calibrators in biological matrices for clinical diagnostic applications requires reference measurement procedures with high accuracy and sensitivity. Testosterone presents a unique challenge with regards to the wide range of endogenous levels across female, male and age based patient populations. We wish to present our efforts towards validating a method for quantitation of Testosterone in calibrators from 0.020 ng/mL to 20 ng/mL in serum by modifying a valid reference measurement procedure. The 1000 fold range of these calibrators in serum presents unique challenges towards designing a method that will accurately measure Testosterone. Data presented will address various elements of the method validation such as precision and accuracy, linearity, limit of quantitation, recovery, matrix effects, extract stability, tracebility. |
Long Abstract
Introduction: Testosterone is measured in male, female and age based populations to diagnose various disease states such as hypogonadism in males and polycystic ovary syndrome.in females. Current state of the art methods use Isotope Dilution Liquid Chromatography Mass Spectrometry (ID-LC MS/MS) to quantitate testosterone in serum. The key advantages to these methods are analyte specificity and selectivity. These ID-LC-MS/MS methods require accurate calibrators to provide accurate results in patient samples.
Therefore, development of accuracy based calibrators in biological matrices for clinical diagnostic applications require reference measurement procedures(RMP) with high accuracy and sensitivity.
These reference measurement procedures are very different from routine clinical methods with regards to accuracy, sensitivity, linearity, robustness and must also have elements of tracebility and commutability.
Method: The method to be presented has two components. The first component is the creation of three Value Assignment Calibrator Curves (Low, Medium and High Curves) that are used to quantitate testosterone in serum calibrators. The second component involves addition of a known amount of internal standard (13C3 Testosterone), and is followed by an elaborate multi-step liquid-liquid extraction of testosterone from the serum calibrators. Extraction is followed by reconstitution of the extract into solvent such the testosterone maybe measured against a particular VAC. . Since the range of the entire matrix calibrator range is 1000 fold, they are divided into three groups for analysis. The low level matrix calibrators are analyzed against the low level Value Assignment Curve (VAC), the mid level matrix calibrators are analyzed against the mid level VAC and the high level matrix calibrators are analyzed against the high level VAC. The same extraction procedure is used to measure residual endogenous testosterone in stripped serum that is used to manufacture these calibrators. The measurement of residual endogenous testosterone is important as this allows us to manufacture matrix calibrators containing exact known amounts of testosterone, in the range of 0.020 to 20.0 ng/mL. NIST SRM-971 is used in each case a Quality Control Sample to verify the accuracy and bias of the method. The performance of this method was tested across multiple days and two analysts with regards to precision and accuracy. Additional elements of validation that were incorporated are matrix effects, analyte recovery, batch size test and extract stability.
Results: Across several days the method was found to be linear for all three Value assignment curves (VACs).
The NIST SRM-971 used as quality control samples were well within 5% from the concentration of testosterone stated in the NIST Certificate of Analysis. Similar results were found for the matrix calibrators at 0.020, 0.040, 0.350, 1.50 and 20.0 ng/mL respectively. The matrix factor was measured and found to be close to 1.0 for all the three levels of VAC. The measured recovery for testosterone and its internal standard was found to be near 100% for all three levels of the VAC. Extract stability was established for seven days and a batch size of 180 injections was ascertained for this method.
Conclusions: Existing Primary Reference Measurement Procedures for Testosterone were taken and modified. The modified method was validated to extend its range from 0.020 ng/mL to 20.0 ng/mL as well as for endogenous measurement of residual testosterone. The method was found to be rugged and suitable for accurately measuring testosterone in serum.
References & Acknowledgements:
1) Susan Tai et al., Anal Bioanal Chem (2007) 388:1087–1094
2) Vesper et al., Clinical Chemistry 59:2372–380 (2013)
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no | Cerilliant |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |