Quantification of Rivaroxaban in Serum Sample

Authors: Mégane Moreau, Serge Auger, Pierre Picard and Jean Lacoursière
Themes: High-Throughput, Anticoagulant, Rivaroxaban, Serum, Luxon-MS/MS
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Rivaroxaban is an anticoagulant used to prevent thromboembolism. Prompt quantification of rivaroxaban in serum is important to avoid overdoses and to adjust their medication. Rapid quantification of rivaroxaban in a patient may become critical in some situations. Here are some examples of those situations from Baglin and al. 2012:

To increase the analysis throughput of the optimization and the follow-up on medications, the Luxon Ion Source® coupled to tandem mass spectrometry (MS/MS) was used.

Our goal for this application note is to present a rapid drug monitoring method in patient serum in 13.2 seconds per sample. To develop this application, we focused on performing a quick and simple sample preparation and analysis.

Sample Preparation Method

Automated Sample Extraction

Serum samples were transferred into barcoded tubes, readable by the Azeo Liquid Handler and were transferred into the system. Each barcoded vial was scanned by the Azeo Liquid Handler (Figure 3).


Samples were extracted as follows:


Figure 3 – Automated extraction system


Table 1 shows the calibration range used for the quantification of Rivaroxaban in serum.

Table 1 – Calibration Range


Drug Calibration Range
Rivaroxaban 4 – 500 ng/mL


LDTD®-MS/MS Parameters


Model: Luxon SH-960, Phytronix

Carrier gas: 6 L/min (air)

Laser pattern:


Figure 4 shows the laser pattern used during these analyses.

Figure 4 – Laser pattern



MS model: LCMS-8060, Shimadzu

Scan Time: 25 msec each transition

Total run time: 13.2 seconds sample to sample

Ionization: APCI (positive)

Analysis Method: MRM mode


Table 2 shows the mass spectrometer transitions in the positive ionization mode for the drug and its internal standard (IS).


Table 2 – Mass spectrometer transitions (Positive)

Drug Transition CE
Rivaroxaban 436.1 → 144.9 30
Rivaroxaban-d4 (IS) 440.1 → 144.9 30


Results and Discussion


Data preparation process

Data acquisition systems of mass spectrometers were not designed to deal with few seconds per sample signal. The synchronization sequence adds 1 to more than 15 seconds between each sample. To bypass this, all samples are acquired in a single file (Figure 5). To allow the analysis of such data, Cascade™ software is designed to detect, split, and integrate every sample peak acquired in a single file.


Figure 5 – Single file mass spectrometer data for 80 samples. A) Rivaroxaban transition. B) Internal standard transition.


Desorption peak

Figure 6 shows a zoomed area of the transition of the internal standard in the single-file window. A speed of 13.2 seconds per sample was reached with sufficient separation for adequate integration.

Figure 6 – Peak to peak timing


Validation Test

Calibration curves ranging from 4 to 500 ng/mL and QCs (Low, Medium and High) were prepared in serum. Replicate extractions were deposited onto a LazWell™ plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.



The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:



Figure 7 shows a typical calibration curve result for Rivaroxaban.


Figure 7 – Rivaroxaban calibration curve


Precision and Accuracy

For the accuracy and precision evaluation, the following acceptance criteria were used:


For the intra-run precision and accuracy experiment, each fortified sample set was analyzed in six replicates. Table 3 shows the intra-run precision and accuracy results for Rivaroxaban. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value.


Table 3 – Intra-Run Precision and Accuracy of Rivaroxaban

Rivaroxaban LLOQ QC-L QC-M QC-H ULOQ
Conc (ng/mL) 4 12 150 350 500
N 6 6 6 6 6
Mean (ng/mL) 4.1 12.1 148.5 357.7 488.5
%CV 10.9 7.7 5.0 7.7 7.0
%Nom 102.5 101.1 99.0 102.2 97.7


For the inter-run precision and accuracy, each fortified sample set were analyzed in five different runs. Table 4 shows the inter-run results. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value.


Table 4 – Inter-Run Precision and Accuracy of Rivaroxaban

Rivaroxaban QC-L QC-M QC-H
Conc (ng/ml) 12 150 350
N 30 30 30
Mean (ng/mL) 12.1 148.5 357.7
SD 0.93 7.44 27.63
%CV 7.7 5.0 7.7
%Nom 101.1 99.0 102.2



Luxon Ion Source® combined to a Shimadzu LCMS-8060 mass spectrometer system allows fast analysis (13.2 seconds per sample) of Rivaroxaban in serum using a simple and automated sample preparation method.