UPLC-MS/MS Bioanalytical Method Validation of Acebutolol and Pindolol using an Analogue Internal Standard

This application note shows the partial validation of a bioanalytical method for acebutolol and pindolol in human plasma using nadolol as an analogue internal standard.

Beta-blockers are a common class of drugs used to treat conditions such as high blood pressure, tachycardia, and cardiac arrhythmia. In this application note, we show the partial validation of a bioanalytical method for acebutolol and pindolol in human plasma using nadolol as an analogue internal standard (Figure 1). The validation was carried out according to the guidelines in the FDA Guidance for Industry on Bioanalytical Method Validation.

Through this experiment, we aim to show that the Waters UltraPerformance LC System combined with the Waters Micromass Quattro Premier XE Mass Spectrometer (UPLC-MS/MS) operating in MRM mode is an accurate, precise, and robust technique which will also yield the benefits of greater speed, sensitivity and resolution over HPLC-MS/MS.

During this experiment we performed a comparison between HPLC and UPLC using a protein precipitation (PPT) sample preparation method.

Protein Precipitation Method

1. 200 μL plasma was spiked with: 
   - 50 μL IS (1.0 μg/mL in water) 
   - 50 μL spike solution (from 0.8 ng/mL–600 ng/mL in water) 
   - When the IS and/or spike solution was not required, the appropriate volume of water was added
2. 600 μL acetonitrile was added to crash proteins
3. Centrifuged at 13,000 rpm for 5 minutes 
4. 200 μL of supernatant diluted with 800 μL water prior to injection

Standard curves and QC samples were prepared as described and shown in Table 1. Three separately prepared validation batches were prepared by protein precipitation and run using UPLC-MS/MS. A standard curve prepared by protein precipitation in human plasma was run using HPLC-MS/MS for comparison.

A validation batch consisted of the following:

• 2 separately prepared calibration curves
• 6 individually prepared replicates of each QC concentration point
• A blank and double blank before each curve
• 2 carryover blanks after each curve

The HPLC, UPLC and MS Conditions used are as follows:

The “Curve” setting in the above gradient tables refers to the gradient profile; adjusting the method to a non-linear curve setting can help separate close running peaks under some circumstances. A graphical representation of the gradient used for this analysis is shown in Figure 2.

All of the calibration standards run by UPLC-MS/MS generated calibration curves with a coefficient of calibration (R²) greater than 0.996. The HPLC-MS/MS run generated calibration curves where R² was greater than 0.997. Typical examples of calibration curves for pindolol and acebutolol (using UPLC-MS/MS) are shown in Figure 3.

Inter-batch calibration statistics are shown in Tables 2 and 3. The statistics for the standard injections are based on 2 replicate injections of the 9 calibration points for each of the 3 inter-day batches. All calibration points show <8% CV with accuracy values between 93.6% –103.7% for both pindolol and acebutolol.

Statistics for the QC injections, shown in Tables 4 and 5, are based on single injections of 6 individually spiked QC solutions at each concentration, for each of the 3 inter-day batches. Both pindolol and acebutolol show <15% CV for the lower limit of quantitation (LLOQ) with <10% CV for the remainder of the quality control standards. Inter-batch accuracy values were observed between 93.2% –111.99% for both pindolol and acebutolol.

FDA guidelines recommend that samples at the LLOQ should have less than 20% CV and deviation from the standard curve. All other unknowns, calibration standards, and QC standards should be within 15%, accuracy values should be within 80–120% at LLOQ, and 85–115% for other standards. 

All of the results generated during the validation of this method comply with and exceed the guidelines set forth by the FDA.

HPLC versus UPLC

In Figure 4, we can see that we get a 3.8 fold increase in signal-to-noise by using UPLC versus HPLC methodology. As well as increases in signal-to-noise and limit of detection, there is also an increase in resolution, giving a better chance of separating the analyte from endogenous peaks. A 2 fold decrease in run time was also observed, meaning that a validation batch was run in only 2 hours by UPLC compared to 4 hours when run by HPLC. An example of both an HPLC and a UPLC chromatogram are shown below for comparison.

We have successfully produced a validated UPLC-MS/MS method for the analysis of pindolol and acebutololin human plasma over the range of 0.2–150 ng/mL. Statistics for accuracy and precision were within the FDA guidelines for bioanalytical method validation. The data generated by UPLC-MS/MS were comparable to that generated by HPLC-MS/MS, however, it was shown that by using UPLC, a 4 fold increase in signal-to-noise ratio for the LLOQ, a 2 fold decrease in run time, and an increase in resolution was achieved. This equates to doubling the throughput of this method, as well as enabling the acquisition of meaningful data for lower sample concentrations. This has several benefits, for example, as it would allow more accurate measurement of the lower part of the PK curve.