Joyce Flanagan (Presenter)
Marshfield Clinic
Authorship: Dale Whipple, Joyce Flanagan
Marshfield Clinic
| Short Abstract Our aim was to develop and validate an automated front-end extraction process for vitamin D including an improved LC/MS/MS method. Complete automation was achieved with the Tecan AC extraction plate on a Tecan EVO 100 system, whereas 25-OH Vit D3 and 3-epi-25-OH Vit D3 were chromatographically separated with a Phenomenex, Kinetex, 2.6 µ pentfluorophenyl (F5) column on a Waters Acquity UPLC Xevo TQ-S LC/MS/MS system. We demonstrated improved assay efficiency, accuracy, and turn-around-time by reducing the sample prep time from a 2.5 hour manual extraction to a 45 min hands-free process and analytical time from 8 to 4 minutes per sample. |
Long Abstract
Introduction: Our existing LC MS/MS method for vitamin D (25-OH Vit D2 and 25-OH Vit D3) assay, validated in 2008 using a manual liquid-liquid extraction for sample preparation that took 2.5 hours of bench time and required multiple manual pipetting steps. Due to our aging technical staff, this method, unfortunately, resulted in a couple of carpal tunnel injury claims over the years. In addition, the LC MS/MS analysis took 8 minutes for each injection and could not separate 3-epi-25-OH Vit D3 (3-epi-D3) from 25-OH Vit D3 (D3), which may result in overestimation of D3. It is known that some pediatric patients have higher percentage of 3-epi-D3. To prevent under-reporting of low D3 results, our policy was to send 25-OH Vit D requisitions for patients under the age of one to a reference lab that claims to be able to separate 3-epi D3 from D3. We sought to develop and validate a new LC MS/MS 25-OH-Vit D method to 1) automate the pre-analytical extraction process, 2) improve chromatographic separation of D3 and 3-epi-D3, and 3) improve the accuracy, precision, and turn-around-time of the assay.
Method: 25-OH Vit D2 and D3 were extracted from serum using the Tecan 96-well AC extraction plate that employs the Tecan Immobolized Coating Extraction (TICE) technology on a Tecan Freedom EVO 100 liquid handler robotic system. A complete walk-away process is achieved by a script programed to pipette the extraction buffer, a carbonated buffer that contains the deuterated stable isotopes for D2 and D3, to the AC extraction plate, followed by the introduction of calibrators, controls, and samples. The mixture is then shaken for 10 minutes on an on-board orbital shaker (Te-shake) where the extraction plate is situated. The extraction buffer is then removed to a waste container. The wash solution is then added, shaken for 2 minutes, and aspirated to the waste container. The elution solvent is added to the extraction plate and shaken for 5 more minutes. The elution solvent is then pipetted from the extraction plate into a 96-well collection plate, which can be directly sampled and injected into a Waters Acquity UPLC equipped with a Phenomenex, Kinetex 2.6 µ pentfluorophenyl (F5) column coupled with a Xevo TQ-S tandem mass spectrometer system using a positive electrospray MRM mode for 25-hydroxy vitamin D analysis.
Results: The extraction process was successfully automated with the Tecan AC extraction plate on a Tecan EVO 100 liquid handling system. Chromatographic separation of 3-epi-D3 from D3 was accomplished with a Phenomenex, Kinetex 2.6 µ pentfluorophenyl (F5) column on a Waters Acquity UPLC system. The analytical time was improved from 8 to 4 minutes with a Waters Xevo LC MS/MS platform.
The performance characteristics of this method are as follows: For 25-OH Vit D2 and D3, respectively; 1) Precision (%CV): intra-assay, 2.09 and 1.77; inter-assay: 4.91 and 4.42; 2) linearity (ng/mL): 400 and 300; 3) LOQ/LOD (ng/mL): 4 and 1 for both; 4) Correlation between the current method: D2=0.94x-0.91 (N=42) and D3=0.93X-1.46 (N=43); 5) Recovery study by spiking: D2 (96.3%), D3 (73.7%); 6) Signal suppression study was performed using the Matuszewski protocol. The matrix effect was observed for both D2 (23%) and D3 (34%). With the addition of isotopic internal standards, the relative signal suppression was estimated to be 4.4%, and 2.8% for D2 and D3, respectively; 7) Sample type study: RTT (red top serum), SST (gel separator serum), LTT(EDTA plasma), GTT (sodium heparin plasma), and PST (lithium heparin plasma with gel separator) were studied and found to be acceptable for routine analysis; 8) Stability study: at ambient temperature, heparinized plasma samples, GTT and PST were stable up to 24 hours, whereas the LTT and RTT were stable up to 72 hours. All sample types were stable refrigerated up to two weeks, frozen up to two month, and through four freeze/thaw cycles; 9) Interference study: Eighty six compounds including amino acids, catecholamines, corticosteroids, vitamins, drugs of abuse, tobacco alkaloids, and common prescription drugs at higher than normal concentrations were spiked to study samples. None of the compounds tested showed any significant interference.
Conclusions: We successfully validated a robust clinical method for 25-hydroxy vitamin D using a novel extraction plate with a robotic liquid handling system sand a F5 column on a new LC MSMS system to achieve our goals of 1) total front-end automation, 2) complete separation of 3-epi D3 and D3, and 3) improved accuracy, precision, and turn-around-time of the assay, and 4) most importantly, health and safety of lab personnel.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | yes | From Tecan for Dale Whipple if abstract is accepted |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |