Category: Automation and High-Throughput Technologies

1166-B - Automated Calibration Curve Preparation and High Throughput Analysis Using LDTD-MS/MS System

Monday, February 5, 2018
5:00 PM - 6:00 PM

Analytical laboratories must begin all their sample analysis by preparing calibration curves. Large volumes of every given concentration are produced and transferred into smaller vials. Each calibration curve aliquot is then stored separately and this tedious process is generally done manually. Using a CERTUS FLEX liquid dispenser, an automated procedure for the preparation of the calibration curve and aliquots is instead established. Three solutions are used: a stock solution of the standard, a dilution solution and a matrix solution. The dispensing system is then set to a specific delivery volume for the preparation of the blank and the 8 calibration standards directly into 2 mL aliquot vials. After the calibration curve preparation, the blank and the standards are extracted and analyzed using an LDTD-MS/MS system.


Three solutions are added on the CERTUS FLEX system: a matrix solution (negative human urine), a dilution solution (mixture of Methanol: Water, 75:25) and a stock solution (10 µg/mL Cocaine in the dilution solution). A rack containing 54 vials (2 mL) is used as the aliquots container. In each vial, the liquid dispenser system adds 900 µL of matrix as well as 100 µL of different ratios of diluent solution/stock solution. One blank and 8 calibration curve standards are prepared (calibration range: 10 ng/mL to 1000 ng/mL). In a single run, six calibration curves (one blank and 8 standards each) are automatically prepared and aliquoted. Each calibration curve is extracted using a liquid-liquid extraction and analyzed using a High Throughput LDTD-MS/MS system.


The liquid-liquid extraction of the calibration curve aliquot is prepared as follows: 100µL of a urine-spiked sample are mixed with 10 µL of Internal standard solution and 100 µL of phosphate buffer. Then, 400 µL of Hexane:Ethyl acetate (1:1) are added, mixed and 2 µL of the supernatant are placed on a LazWell plate and evaporated to complete dryness. Samples are desorbed using a laser power ramp of 0 to 45% in 6 seconds. The mass spectrometer is operated in positive MRM mode. The transition 304 → 182 was used for Cocaine and 307 → 185 for Cocaine-d3. The linearity, slope and accuracy of each calibration curve preparation are evaluated. The interference of the blank at the lowest standard is verified.


With the aforementioned system used, the linearity of the calibration curves attains values greater than R2> 0.996. Slope values range within 0.0069 and 0.0071, showing a high consistency between each preparation. Back-calculated concentration of each calibration point is evaluated and accuracies between 86.5% and 112.3% are obtained. Blank interference is between 0.0% to 13.9% at the LLOQ and 0.0% to 0.6% for the internal standard.

Jean Lacoursiere

Phytronix Technologies
Quebec, Quebec, Canada

President and Owner of Phytronix Technologies.